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Journal of Molecular Neuroscience Copyright © 2005 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN0895-8696/05/26:133–154/$30.00 DOI:10.385/JMN/26:02:133

ORIGINAL ARTICLE

How Proteins Come Together in the Plasma Membrane and Function in Macromolecular Assemblies Focus on Receptor Mosaics

Luigi F. Agnati,*,1 Diego Guidolin,2 Susanna Genedani,3 Sergi Ferré,4 Albertino Bigiani,1 Amina S. Woods,4 and Kjell Fuxe5 Department of Biomedical Sciences, Sections of 1Physiology and 3Pharmacology, University of Modena and Reggio Emilia, 41100 Modena, Italy; 2Department of Human Anatomy and Physiology, Section of Anatomy, University of Padua, 35100 Padua, Italy; 4National Institute on Drug Abuse, Department of Health and Human Services, Intramural Research Program. Baltimore, MD 21224; and 5Department of Neurosciences, Karolinska Institute, SE-17177 Stockholm, Sweden

Abstract Some theoretical aspects on structure and function of proteins have been discussed previously. Proteins form multimeric complexes, as they have the capability of binding other proteins (Lego property) resulting in multimeric complexes capable of emergent functions. Multimeric proteins might have either a genomic or a postgenomic origin. Proteins spanning the plasma membrane have been analyzed by considering the effects of the microenvironment in which the protein is embedded. In particular, the different effects of the hydrophilic (extracellular and intracellular) versus the lipophilic (intramembrane) environment have been considered. These aspects have been discussed in the framework of membrane microdomains, in particular, the so-called rafts. In α-helix proteins the individual peptide dipoles align to produce a macrodipole crossing the entire membrane. This macrodipole has its positive (extracellular) pole at the N-terminal end of the helix and its negative (intracellular) pole at the C-terminal end. This arrangement has been analyzed in the framework of the counter-ion atmosphere, that is, the formation of a cloud of small ions bearing an opposite charge. Excitable cells reverse their resting potential during the all-or-none action potentials. Hence, the extracellular side of the plasma membrane becomes negative with respect to the intracellular side. This change of polarization affects also the direction and magnitude of the α-helix dipole in view of the fact that there is a displacement of the counter ions. The oscillation in the intensity of the dipole caused by the action potentials opens the possibility of an interaction among dipoles by electromagnetic waves. DOI:10.1385/JMN/26:02:133 Index Entries: Protein–protein interactions; Lego property; electrostatic interactions; hydrogen bonds; van der Waals forces; lipophilic groups; lipophobic groups; receptor mosaics; electromagnetic waves.

Introduction Some theoretical aspects on structure and function of proteins are discussed here; particularly, the possibility that multimeric proteins have different

biological purposes in relation to their genomic or postgenomic origin. The first basic aspect to be dealt with is the evidence that some multimeric proteins are encoded in the genome as such, whereas others are assembled after the synthesis of the protein building

*Author to whom all correspondence and reprint requests should be addressed. E-mail: [email protected] Dedicated to Professor Paolo Marrama, former Chair of Endocrinology in Modena.

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134 blocks. An important framework for this discussion can be found in the Lego property (Agnati et al., 2002, 2004a), which is deeply linked to the structural analysis of domains and motifs allowing protein-protein interactions. Proteins buried or spanning the plasma membrane are analyzed here by considering the effects of the microenvironment in which the protein (or parts of the protein) is embedded. In particular, the different effects of the hydrophilic (extracellular and intracellular) versus the lipophilic (intramembrane) environment are considered. These aspects are discussed in the framework of membrane microdomains (Agnati et al., 1990), particularly lipid rafts (LRs) (Simons and Ikonen, 1997); in addition to protein–protein interactions, the relevance of lipid–lipid and protein–lipid interactions also are considered, although the final evidence for raft existence remains to be obtained (Munro, 2003). All of these interactions are of paramount importance for molecular networks and, in particular, for interactions occurring at the plasma membrane level within the horizontal molecular networks (HMNs) (Agnati et al., 2002, 2003a, 2004a, 2004b). The concepts involving protein–protein interactions are applied to the phenomenon of receptor–receptor interactions ([RRIs]; Agnati et al., 1980, 1983, 2003b; Fuxe et al., 1983; Fuxe and Agnati, 1985, 1987), the structural result being the formation of functioning receptor mosaics (RMs) (Agnati et al., 1982, 2002, 2004a). In this paper we advance the hypothesis that RMs might work as macromolecular assemblies in the core of the structural organization and integrative operation of the HMNs.

Lego Property Proteins, carbohydrates, and lipids form molecular networks at cellular and extracellular levels. Proteins are the main constitutive elements of molecular networks, as proteins possess the Lego property, that is, the capability of a stereochemical interaction with regard to other proteins resulting in high-molecular-weight complexes that are capable of emergent functions. As discussed previously (Agnati et al., 2004a, 2004b), we have differentiated the Lego property from the allosteric effects of certain functional proteins. Allosteric control refers to the existence on a protein (classically an enzyme or, more generally, a protein operator) of one or more secondary binding sites that can bind regulators. The binding of a regulator to the protein can enhance


Agnati et al. (positive allosteric regulator) or reduce (negative allosteric regulator) the affinity of the primary binding site for its ligand. However, it should be noted that the allosteric modulation of proteins can be of paramount importance in making possible the fitting process between two protein modules. Thus, allosteric modulation can represent a preliminary and necessary step for the appearance of the Lego property and thus of protein complexes endowed with emergent characteristics. In this context it is of relevance to mention the fact that some molecular interactions are only formed among proteins that have undergone a conformational change, for example, after the phosphorylation-dephosphorylation process (Greengard et al., 1998). The Lego property is therefore the ability of proteins to specifically interact with other proteins. These interactions can occur both in the plane of the membrane (HMNs) or vertically (vertical molecular networks), that is, toward the extracellular side and/or toward the intracellular side of the cell. The relevance of the Lego property is indirectly supported by the fact that the human genome contains fewer genes (approx 35K) than predicted previously (approx 70K). It should be taken into consideration that the real number of the various proteins derived from a given gene is expanded by the mechanism of alternative splicing. However, the dilemma of the many different functions to be performed by a limited array of proteins remains. In part, it can be solved by considering that the functional repertoire of an array of proteins is enhanced by the formation of protein complexes via protein–protein interactions. The idea is based on the principle that underlies the construction of complex molecules via the assembly of simple molecules, such as the synthesis of a huge variety of proteins from only 20 different amino acids. The functional repertoire of proteins is further enhanced by the formation of complexes with other types of molecules such as lipids and carbohydrates. Furthermore, as pointed out by Petsko and Ringe (2004), “an additional explanation would be, that most likely in multi-cellular organisms, a given protein depending on its surrounding or location could have more than one distinct biochemical and/or cellular function. The biochemical functions may include catalysis, binding, participation as a structural molecule in an assembly, or operation as a molecular switch. The extent of this functional diversity for any one protein is only just beginning to be appreciated.”

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Protein Interactions: Receptor Mosaics These aspects are analyzed starting from proteinprotein interactions (i.e., of the Lego property). The following definitions are of basic importance for the analysis of this property: 1.

2.

Domain (or module): a compact unit of protein structure that is usually capable of folding stably as an independent entity in solution. Hydrophobic cores appear to be essential for the stability of domains. Structural motif: a characteristic amino acid sequence that might comprise a whole domain but usually consists of a small local arrangement in secondary structure elements, which coalesce to form a domain. A structural motif can be associated with a biochemical function (functional motif).

Some proteins are composed of a single structural domain; however, most proteins are built up in a modular fashion from two or more domains that are fused together (Lego property of proteins). Thus, the Lego property leads to the formation of protein oligomers (or protein multimers). The term oligomerization is used in molecular biology to indicate any multimeric protein that contains a finite, relatively small number of identical or similar subunits (i.e., protomers) that are not held together by a covalent linkage but, rather, through noncovalent interactions that are known to be in a flux of associationdissociation, which is influenced by changes in the milieu where the oligomers are immersed. It should be emphasized that the term oligomerization for the formation of protein complexes does not indicate the underlying mechanisms promoting the formation of oligomers, as these could take place because of hydrophobic or electrostatic interactions, hydrogen bonds, or a combination of all of the above. At least two types of Lego properties should be considered: one transient and one permanent. Several domains can be recognized in proteins; and during evolution, these domains (secondary structure elements) can be linked together by means of loops that join them and are usually located on the surface of the tertiary structure; they make few, if any, contacts with the rest of the connecting domains. This can be thought of as a permanent type of Lego property (genome-cabled protein–protein interaction) to be differentiated from the transient Lego property that occurs when proteins synthesized as such interact to form plastic macromolecular complexes capable of fulfilling a certain metabolic function. Thus, two types of Lego property can be recognized: 1.

A stiff one: the genomic Lego property is used by evolution to construct, by single domains, a stable tertiary structure, that is, a fixed precabled biochemical


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2.

machinery. The covalent bonds assure the stability of the complex. Aflexible one: the postgenomic Lego property is used as needed by the cell metabolism, using single domains to construct, mainly through noncovalent bonds, a flexible tertiary structure. Thus, a plastic, transient biochemical machinery is formed, which the cell employs to carry out a certain metabolic task. It might be important to distinguish the postgenomic assembling of protein modules carried out at the level of the endoplasmic reticulum (ER), the Golgi apparatus (GA), and at the final destination, that is, the plasma membrane.

Some aspects of the two types of Lego property are illustrated in Fig. 1, where it is shown that a multimeric protein can be obtained via fusion of genes that once code for separate proteins or via interaction of different domains. Obviously, whereas the first type of Lego leads basically to a single permanently cabled macromolecular complex, the second type leads to a set of transiently cabled macromolecular complexes. Thus, the two types of Lego are suited for different tasks. In the first case, the species has found that a certain macromolecular complex is of such importance for cell function that it is worthwhile to have it coded in the genome. In the second case, the importance is in the entire set of macromolecules that can be flexibly cabled according to the needs. As mentioned above, this second type of domain assembly can occur either at the ER or at the GA or, for example, at the plasma membrane level, and these different possibilities are probably related to different functional needs. Another important aspect to be considered and that further underlines the different flexibility of the genomic versus the postgenomic protein domain assemblies is the multiple conformational states that a protein can assume, that is, its energy landscape. Proteins can assume a large number of slightly different structures, each with potentially different biochemical characteristics (Frauenfelder et al., 1991). Abasic feature to be considered is therefore the energy landscape, as this can describe the range of possible conformations that a protein can assume and the relative stability of each conformation (Kauffman, 1993; Agnati et al., 2002). It is very likely that, in vivo, only a restricted number of conformations are allowed, as the various chemicophysical influences impinging on proteins are strictly controlled by the homeostasis of the internal milieu, especially of the brain. However, a variety of possible protein conformations should be considered.

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Fig. 1. Schematic representation of the two types of Lego properties: the genomic Lego property and the postgenomic Lego property. The first one is used by evolution to construct a stable tertiary structure by single protein domains; the second one is used as needed by the cell metabolism to construct a flexible tertiary structure by means of building blocks. It should be noted that the genomic-assembled multimeric protein complex has covalent bonds among the modules, whereas the postgenomic multimeric protein complex is attributable to noncovalent bonds among modules. A basic feature to be considered is the energy landscape of the proteins, i.e., the multiple conformational states they can assume in response to changes in the chemicophysical conditions of the environment in which they are embedded. Thus, while in the genomic assembly these influences affect the multimeric protein as such; those in the postgenomic assembly affect the single modules, increasing enormously the combinatorial possibilities of assemblies and hence the plasticity of the process of assembling the multimeric protein (for further details, see text).

By organizing modules for assembly, the biochemical machinery can take full advantage of the wide spectrum of possible conformations for each module. Hence, the combinatorial possibilities are markedly increased, when compared with the preassembled modules because of the restrictions of genomic coding of the assembly. In the case of the postgenomic assembly, one important aspect is its stability, as links among modules should be stable enough to allow them to operate as a single functional unit, but they should not be irreversible. Furthermore, there must be a threshold to prevent the formation


of nonoptimal complexes, that is, of complexes formed with nonoptimal bonds leading to unwanted protein assemblies. We propose that in some instances, the thermal energy of molecules (approx 2.5 kJ/mol at room temperature) operates as a threshold capable of preventing the formation of nonoptimal complexes as they assemble. The assembly of protein modules is attributable to the sequential formation of different types of atom-atom interactions or of interactions between entire groups of bonded atoms. These interactions are very weak, being hydrogen bonds (the strength depends on the charge

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Protein Interactions: Receptor Mosaics of acceptor and donor atoms), electrostatic interactions (the strength depends on distance and markedly on the dielectric constant of the medium; see below), and van der Waal’s forces (the strength depends markedly on the distance, which falls rapidly beyond 4 Å of separation). All of these interactions have an energy that is not so much different from the average thermal energy of molecules. Hence, if two protein modules approach each other in a nonoptimal way or two protein modules that are not supposed to interact are initiating the interaction process, the thermal threshold can stop the process by disrupting the few weak bonds formed. One could also surmise that in homeothermic animals (as humans are), in view of the high temperature (especially of the brain [Yablonskiy et al., 2000]), the threshold preventing unwanted protein complexes is the highest, therefore resulting in a higher safety factor. Stability in the complexes is reached when hundreds of these weak interactions occur, and this happens only when there is a high complementarity between modules, that is, when a large number of electrostatic interactions are possible. The hydrogenbond donors and acceptors at the interface between the two monomers match with each other, the interfaces having reached such a distance that van der Waal’s contacts are possible. It should also be considered that disturbances in the Lego property can cause pathologies. It is suggested that protein conformational disorders (PCDs) have as a common hallmark the misfolding of an otherwise normal protein (Thomas et al., 1995; Carrell and Lomas, 1997; Dobson, 1999). These misfoldings lead to abnormal polymerization of the protein monomers (i.e., to formation of toxic protein supramolecular complexes) (Lee et al., 2004; Recchia et al., 2004). That is, the protein misfolding favors the appearance of abnormal protein-protein interactions; hence, the Lego property, not properly directed, can cause PCDs. Pathologies belonging to PCDs have been described at the CNS level (e.g., Alzheimer’s disease, transmissible spongiform encephalopathies, Parkinson’s disease) (Lee et al., 2004). However, it should be considered whether—even if to date they are not yet demonstrated—PCDs can occur also at the peripheral level. It should be noted that the ubiquitin-proteosome system is responsible for the clearance in cells of most soluble proteins, especially of short-lived regulatory proteins and damaged and misfolded proteins


137 (Hernández et al., 2004). Thus, the ubiquitin– proteosome system can be considered, inter alia, as a control system of the Lego property and, hence, as a system capable of reducing the risk of inappropriate protein–protein interactions.

Theoretical Considerations on Protein Interaction Domains Protein interaction domains are independently folded modules, 35–150 residues in length, that recognize another protein (or, more generally, a ligand) via a specific recognition motif, which could be as little as two residues, or a post-translational modification such as phosphorylation (Woods, 2004). Usually they can still bind their target partner (or ligand) even if expressed independently of their host protein. Interaction domains show a certain specificity, that is, they preferentially bind some motifs. Thus, the hypothesis is put forward that there is a code made by motifs. This is obviously a degenerate code, as more than one ligand can be recognized by one and the same motif. However, the specificity of the recognition process between the two partners, for example, a ligand and the protein or two proteins carrying the appropriate interaction domains, can be achieved by various mechanisms (see Fig. 2): 1. 2.

3.

The timing of the expression. The two partners of the interaction are expressed in a time interval that allows them to interact. The same cellular locations of the possible partners for the protein carrying the interaction domain that recognizes them. Usually, there are mechanisms targeting the protein and the appropriate partner (i.e., the two partners) to the same compartment. The modulatory actions (i.e., allosteric modulation) of ligands to mask/unmask the binding motif only on the two appropriate partners.

The cooperation of these mechanisms allows specificity to the degenerate code of motifs. This is an important issue, as it is very likely that when the proper contribution of one of these mechanisms is defective there is loss of specificity in the recognition process between the two partners and unwanted macromolecular complexes can be formed that potentially can cause metabolic disorders. It can also be surmised that the three mechanisms help the understanding of one important feature of some proteins, especially in multicellular organisms, that is, the functional diversity for one and the same

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Fig. 2. A Venn’s diagram of the mechanisms allowing selectivity in the formation of supramolecular complexes from single protein domains using the postgenomic property of proteins (for further details, see text).

protein—the existence of multifunctional proteins (Petsko and Ringe, 2004; see also above). According to the time of expression, the cellular compartment location, and the modulatory actions of ligands (or tags), a protein can fulfill a different task (or have a different fate). In this regard it should be considered that all three mechanisms are fully in operation only in eukaryotic cells in which the timing of expression of a protein is regulated much more strictly than in prokaryotic cells. The number of cellular compartments is also more numerous, as prokaryotic cells lack organelles and cytoskeleton structures. The


fate of a protein is controlled more accurately, as exemplified by the complexity of the proteolytic machinery responsible for targeted protein degradation. Proteins targeted for destruction are fed into the multiprotein complex called the proteosome (see also above). In prokaryotes, proteosomes consist only of a tunnel-like enzymatic core. In eukaryotes, they have additional caps that recognize and bind only the proteins carrying an appropriate signal for destruction. Some general aspects of protein-protein interactions can be given (Borgan and Thorn, 1998; Kortemme and Baker, 2002):

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Fig. 3. Schematic representation of lipophobic effects, which allow protein-protein interactions if they have polar groups buried in the membrane. It is suggested that these polar groups in a lipid environment have the tendency to selfassociate and thereby minimize their contact surface area with the nonpolar medium of the internal part of the membrane. In particular, it should be mentioned that electrostatic interactions, as well as hydrogen bonds, are stronger in a medium of low polarity (for further details, see text).

1.

2. 3. 4.

Most interaction interfaces are composed of relatively large protein surfaces (>600 Å2) with complimentary conformations and electrostatic salt bridging for enhanced stability. A small set of hot spot residues at the interfaces contribute significantly to the free energy binding of the protein–protein interaction. Hot spots are clustered mainly at the center of the interfaces. Hot spots are protected from contact with bulk solvent by peripheral residues that do not contribute significantly to the binding energy of the protein–protein interaction. This ring of peripheral residues excludes solvent from the center, where a reduction of the effective dielectric constant strengthens electrostatic and hydrogen bonding interactions.

These concepts can be used to characterize some aspects of the RRIs, as discussed below in Molecular Interactions and Lipid Organization of the Plasma Membrane: Focus on HMNs and RMs.

Possible Relevance of the Lipophobic Effect for Electrostatic Interactions of Protein–Protein Interactions If a protein buried within the membrane (e.g., an α-helix) has amino acids with a polar side chain (hydrophilic groups), these groups can be used for


protein–protein interactions inside the membrane because of the existence of a lipophobic effect (see Fig. 3). Thus, the hypothesis is put forward that besides the well-known hydrophobic effect (i.e., the tendency of nonpolar groups in water to selfassociate and thereby minimize their contact surface area with the polar solvent), a lipophobic effect can take place inside the membrane favoring protein association. That is, polar groups in a lipid environment have the tendency to self-associate and thereby minimize their contact surface area with the nonpolar medium of the internal part of the membrane. In particular, α-helices would assemble into molecular aggregates to shield their polar residues from the hydrophobic environment. Electrostatic interactions (positive–negative charge interactions) have a range that depends on the dielectric constant of the medium, and thus long-range electrostatic interactions can occur inside the membrane. This could be one of the mechanisms allowing proteinprotein interactions inside the membrane. The binding of a ligand to its receptor can cause an allosteric change in the receptor conformation with the potential exposure of polar groups, and this conformational change can favor—because of the existence of the lipophobic effect—protein–protein interactions, as these interactions lead to the self-association of

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Fig. 4. Schematic representation of the extramembrane parts of a GPCR that might represent highly flexible steric structures immersed in a polar medium (water). Hence, they can have weak electrostatic interactions with other GPCRs (and/or proteins). These structures are therefore particularly suited for fishing ligands in the extracellular and intracellular environment, respectively.

the polar groups to minimize their contact area with the lipid environment. In this context, it should be considered that not only electrostatic interactions but also hydrogen bonds could be stronger in a medium of low polarity (Dougherty and Lester, 2001). The lipophobic effect with electrostatic interactions inside the membrane might be at the core of many RRIs. This aspect is further developed in Fig. 4, where it is suggested that the extramembrane parts of a G protein-coupled receptor (GPCR) are highly flexible steric structures immersed in a polar medium (water). Hence, they have weak electrostatic interactions with other GPCRs (and/or proteins), which can be remodeled by thermal energy and alterations


Agnati et al. in the ion activity of the medium. These structures are therefore particularly suited for “fishing” ligands in the extracellular and intracellular medium, respectively. On the other hand, the intramembrane polar groups of the GPCR can interact because of the lipophobic effect and/or take part in strong electrostatic interactions and/or hydrogen bonds in the apolar medium. Thus, they form a stable connection with other GPCRs (and/or membrane proteins) that is of importance in allowing the flexible parts immersed in the watery medium to have stable weak interactions (see above) as they are anchored to a macromolecular complex that serves as a platform. In the α-helix the individual peptide dipoles align to produce a macrodipole crossing the entire membrane (Petsko and Ringe, 2004). This macrodipole has its positive (extracellular) pole at the N-terminal end of the helix and its negative (intracellular) pole at the C-terminal end (Hol, 1985). The magnitude of this helix dipole increases with the length of the helix, provided the cylinder remains straight. Hence, anions are bound at the amino terminus, and cations at the carboxyl terminus of the helix (Petsko and Ringe, 2004). This arrangement can be analyzed in the framework of the counter-ion atmosphere (see, e.g., Mathews and van Holde, 1999), which is the formation of a cloud of small ions bearing an opposite charge. The effect of counter-ions is a shielding of the macrodipoles, which makes negligible their direct electrostatic interaction (Ben-Tal et al., 1997; White and Wimley, 1999), because of the rapid reduction of the electric fields with the distance. The static field from each unitary charge in a salty fluid falls from ~6 × 106 V/m at a range of 1 nm to 0.7 V/m at 10 nm, and at a distance of 500 nm the field is almost null. Excitable cells, however, reverse their resting potential during the all-or-none action potentials. Hence, the extracellular side of the plasma membrane becomes negative with respect to the intracellular side. This change of polarization affects also the direction and magnitude of the α-helix dipole because there is a displacement of the counter-ions. As a consequence of this change, the hypothesis is put forward that each dipole can locally tether opposite charges across the plasma membrane. An increased amount of anions near the positive pole could cause a local release of cations in the neighborhood of the C-terminal end of the helix (negative pole) and vice versa. Thus, it should be considered whether the binding of, for example, Cl– or bicarbonate, to the extracellular amino terminus of the helix can cause the

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Protein Interactions: Receptor Mosaics release of Ca2+ or protons from the intracellular carboxyl terminus to activate signal pathways and, conversely, the binding to the intracellular carboxyl terminus of the helix of, for example, Ca2+ or protons, can cause the release of Cl– or bicarbonate at the extracellular amino terminus (see Fig. 5A). Hence, we suggest the possible existence of a fast, to date unacknowledged, biochemical mechanism for the transfer of a signal across the plasma membrane. Furthermore, the oscillation in the intensity of the dipole caused by the action potentials opens the possibility of an interaction among dipoles and/or with neighboring structures resulting in synchronization mechanisms by which the strength of the electric field could be considerably intensified (see Fig. 5B). It has been estimated (Gutmann, 1992) that a dipole characterized by the oscillation of a unitary charge could make, in a dissipative medium, a ~102 V/m signal at 10 nm from the protein helix with an energy transfer of ~0.004 kT per cycle. It should be mentioned that α-helices tend to associate into bundles once they are inserted into the membrane (Bordi et al., 1997). Therefore, the effects described for a single α-helix can be magnified by bundle arrangements of α-helices, leading to significant fields. These waves could be detected by other dipoles (possibly also on neighboring cell membranes in local circuits), which therefore act as receivers. The analysis of a two-dimensional system of classic dipoles (Bedanov, 1992) revealed that long-range ordered behavior could emerge as a result of such interactions. In this respect, a quantum analysis of systems formed by biological dipoles was proposed by Frohlich (1968). He began with the observation that action potentials induce quite strong fields across the plasma membrane. Typically, potentials of 10–100 mV over distances of 0.01–10 µm are generated, producing local fields in the range of 103–107 V/m. For instance, the 50-mV transmembrane potential needed to open a sodium channel corresponds to an electric field of ~5 × 106 V/m. Fields of this magnitude could provide the condition for the development of coherent elastic and electrical vibrational modes of the whole system (Frohlich, 1975; Pokorny et al., 1984) in the 10- to 100-GHz frequency range. Some experimental evidence consistent with the real occurrence of these processes at the membrane level was provided by Vos et al. (1993). The biological effects of these phenomena, however, should be investigated more thoroughly. Taken altogether, these actions could represent one of the mechanisms for the rapid reshuffling of


141 protein interactions in unit rafts (see below; Pralle et al., 2000) and for the information exchange among microdomains.

Molecular Interactions and Lipid Organization of the Plasma Membrane: Focus on HMNs and RMs Horizontal molecular networks (HMNs) are molecular circuits made by molecules embedded and/or associated with the plasma membrane that carry out complex functions of basic importance for informational exchange between the cell and the extracellular environment. Hence, HMNs are twosided input/output networks, that is, they can receive signals from the extracellular as well as from the intracellular environment of the cell and can give an output signal to both the extracellular and the intracellular environment of the cell. Thus, HMNs work as intelligent interfaces between extracellular and intracellular networks (Agnati et al., 2002, 2003a) and have been indicated as the main target of electronic and chemical signals present at the local circuit level (Agnati et al., 2004b). The concept of an HMN is supported by the demonstration in the plasma membrane of specialized microdomains. Of particular relevance among these are the LRs, which denote dynamic assemblies of cholesterol and sphingolipids scattered within a fluid, disordered phase of the lipid bilayer (Simons and Ikonen, 1997; Munro, 2003). Thus, LRs concentrate special classes of lipids and proteins that function in transmembrane signaling events. Hence, LRs have emerged as specialized liquid-ordered membrane platforms for HMNs specialized for signal integration and transduction. It has also been suggested that different types of LRs can be distinguished on the basis of structural proteins, modifiers of raft function (MORFs [Razani et al., 2002]). Caveolin-1 is a MORF, and when integrated into the macroenvironment of the LR this microdomain invaginates and forms caveolae. Thus, caveolae are considered a specialized type of LR containing the caveolin proteins (Paratcha and Ibanez, 2002). Data have been collected on the possible functional role of caveolins in signal transduction (see Fig. 6A,B). From a general standpoint, functionally different molecules can be recognized in the HMN, such as 1.

Receiving elements (e.g., receptors): molecules capable of recognizing and decoding signals reaching into the HMN from outside of the network itself.

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3.

4.

Structural elements: molecules giving mechanical support to the HMN, for example, by anchoring some elements to the plasma membrane (anchoring proteins) or by linking two proteins (adapter proteins). Transmission elements (e.g., G proteins): molecules capable of transferring a signal from one molecule to another. Often, the received signal is modified and sometimes integrated with other signals. Effector elements: molecules capable of producing an output signal toward the outside of the HMN. In the case of transmitter-gated ion channels, they represent both receiving and effector elements, as the binding of the neurotransmitter opens or closes the ion channel.

However, LRs are not simply platforms for HMNs, but lipids in the LRs have a role both in the structural organization of the HMNs and in the elaboration of the information carried out by the HMNs. In particular, lipid–protein interactions are of importance, as lipids tend to adopt a super-lattice distribution and the phospholipid distribution is affected by the charge of the head groups (Virtanen et al., 1998). Lipid bilayers are stratified structures with a characteristic transbilayer profile in which the hydrophobic core on the average only occupies about one-half of the fluid bilayer thickness (approx 30 Å). On both sides of this core is a heterogeneous chemical layer (approx 5–10 Å) with polar or charged head groups prone to noncovalent binding and, hence, of basic importance for interactions with proteins (Gil et al., 1998). Therefore, the plasma membrane should be analyzed both according to the x–y dimensions (distribution of patches of microdomains) and to the z dimension. Generally, it is possible to propose the scheme of Fig. 7 for aspects on the z dimension. The lipid-mediated protein–protein interactions should also be mentioned (Gil et al., 1998). As shown in Fig. 7, there is a selective accumulation of the lipid species that hydrophobically matches the protein hydrophobic domain at the protein–lipid interface and, hence, wets this interface (lipid annulus). Awetting layer can be shared by two or more proteins giving rise to protein clusters. Finally, not only islands should be considered in the lipid bilayer (rafts) but also fluid regions that could represent preferential pathways for the migration of

143 volume transmission (VT) signals, which in this case are represented by lipophilic molecules such as diacylglycerol (DAG). The energy moving these fluid pathways like a stream (vector of the VT signals) could be mechanical forces (e.g., distortion of the plasma membrane caused by external forces [pressure waves, osmotic effects] or simply insertion of new proteins into the membrane) or even highly localized thermal gradients (e.g., uncoupling protein 2 action). Recently, it has been suggested that the basic unit of a raft can be formed by a single protein molecule surrounded by lipids that are especially suitable, owing to polarity and steric complementarity (Vereb et al., 2003). In the framework of our previous discussion it is important to mention data suggesting that the exact composition and nature of the α-helices will substantially influence how and in which phospholipid environment this polypeptide chain can exist (Lewis et al., 2001). In conclusion, there are probably very strict relationships between the unitary LR and the HMN that uses this LR as a platform, because (1) the lipid environment affects the proteins that can be inserted into the membrane; (2) the proteins inserted in the membrane can be organizing centers for distribution and arrangements of lipids in the raft; (3) the proteins inserted in the membrane can attract and repulse other charged molecules (lipids and proteins) in the raft, allowing a continuous reshuffling of the molecular distribution in the raft; and (4) both lipids and proteins in the LR can have a structural and functional role in the HMN. If these assumptions are correct, it would be of importance to discover the possible matches between proteins–lipids, proteins–proteins, and lipids–lipids and if there are proteins that have a special (pivotal) role in organizing unitary rafts.

GPCRs and Receptor–Receptor Interactions There are at least eight families of GPCRs that show no sequence similarities to each other but are characterized by seven membrane-spanning αhelices and activate a similar set of heterotrimeric G proteins. Homo- and heteromerization of GPCRs seem to be the rule and often an absolute requirement

Fig. 5. Schematic representation of the hypothesis that α-helices can operate as dipoles and as such they can tether opposite charges across the plasma membrane. (A) The actual binding of anions at the positive pole causes the release of cations at the C-terminal end of the helix (negative pole); the binding of cations at the negative pole causes the release of anions at the N-terminal end (positive pole). Hence, we suggest the possible existence of a fast, to date unacknowledged, biochemical mechanism for the transfer of a signal across the plasma membrane (for further details, see text). (B) Oscillations in the intensity of the dipole caused by action potentials can generate electromagnetic waves that can affect conformation and function of neighboring proteins, e.g., ion channels (for further details, see text).


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Fig. 6. (A) Schematic representation of structural, biochemical, and functional relationships between ligands, caveolae, and G-proteins (for review, see Smart et al., 1995). (B) Schematic representation of one of the multiple functional roles of caveolins. Caveolin-1 can inhibit Gαs and PKA activity. Thus, caveolin-1 is a component of a threshold mechanism (1–2), capable of reducing noise because of possible GPCR constitutive activation. Furthermore, caveolin-1 is part of a negative feedback control (FB−) that maintains caveolin-1 expression at its set-point level.

for activation. As mentioned above, this phenomenon originally was postulated and supported by indirect evidence in the work of Agnati and Fuxe (Agnati et al., 1980, 1983, 2003a; Fuxe et al., 1983; Fuxe and


Agnati, 1985, 1987). Thus, it has been demonstrated that RRIs occur at the plasma membrane level, and the functional consequence of these interactions is an integration of the recognition and decoding

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Fig. 7. Schematic representation of aspects of the z dimension of the plasma membrane. The membrane might consist of two 5- to 10-Å-thick external layers (one facing the extracellular fluid and one facing the intracellular fluid), where the polar or charged head-groups of the lipid molecules are concentrated. The inner part of the membrane might be nonpolar and approx 30 Å in thickness. As shown, a selective accumulation of the lipid species that hydrophobically matches the protein hydrophobic domain at the protein–lipid interface forms a kind of lipid annulus at this interface (i.e., in the inner part of the membrane). This lipid wetting layer might be shared by two or more proteins giving rise to protein clusters.

processes carried out by the receptors (for a review, see Fuxe and Agnati, 1987; Agnati et al., 2003a). It is therefore a theoretical and experimental problem of the highest relevance to clarify the biophysical aspects of RRIs and the structural/functional consequences that this interaction causes on the GPCRs of the oligomer. In 1995 it was suggested that RRIs could occur via interactions of extracellular, intramembrane, and intracellular domains of GPCRs (Agnati et al., 1995). Let us apply the theoretical concepts discussed above, in Theoretical Considerations on Protein Interaction Domains, to the RRIs involving GPCRs. The following may be surmised: 1.

The extracellular and intracellular direct RRIs might not be of great importance for the formation of a stable oligomer in view of the small area of the interfaces unless large adapter proteins are involved. In the case of the involvement of large adapter proteins, the existence of strong interaction spots clustered at the center of the interacting surfaces should be demonstrated


2.

3.

together with a peripheral ring of residues for the exclusion of the solvent. The intramembrane RRIs can be of high importance in view of the lipophobic effect and the low dielectric constant and, hence, the very effective electrostatic and hydrogen bonding interactions. The intramembrane RRIs might rely on an interaction surface large enough to account for the stability observed for receptor dimers and oligomers. The αhelices are at least 30 Å (20 residues) long, spanning the thickness of the hydrophobic portion of the lipid bilayer, and approx 5 Å large. From these data it can be deduced that usually more than one helix is involved in the RRI to overcome the minimal size (600 Å2) necessary to achieve a stable interaction.

Because in several cases receptor oligomers are formed before reaching the plasma membrane (Lee et al., 2003a) it might be suggested that in some instances other biochemical mechanisms (e.g., molecules operating as chaperones) can stabilize the oligomer before its insertion into the plasma membrane. Furthermore, it is likely that in most cases

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146 extracellular, intramembrane, and intracellular domains of GPCRs are all involved in RRIs, even if on the basis of the available data the stability of the macromolecular complex might be achieved above all because of interactions among the intramembrane helices and/or the bridging action of adapter proteins. The possibility that adapter proteins can interact also with the extracellular domains of GPCRs to help the stabilization of receptor oligomers should not be overlooked.

GPCRs as a Special Class of Inputs to HMNs Receptors complexes can be recognized in HMNs; and receptors within these complexes can interact with each other via RRI to form a RM and give integrated outputs, as RRIs can generate novel ligand binding (as pointed out in Agnati et al., 1980) and signal transduction properties (Agnati et al., 1982, 1986, 2002, 2003b; Fuxe et al., 1983; Fuxe and Agnati, 1985, 1987; Franco et al., 1996, 2003). The first aspect to be considered in the context of RRIs is the dilemma of whether the agonist promotes dimerization or whether dimers are already preformed. Gouldson et al. (2000) suggests that dimers are preformed and merely rearranged in the presence of the agonist. Actually, experimental facts suggest that dimerization can occur at various levels of the physiological steps, such as at the level of the ER, or possibly the GA, or the plasma membrane (Lee et al., 2003a). There is substantial experimental evidence for this view, as it has been shown that receptors GABABR1 and GABAB-R2 form heterodimers in the ER (White et al., 1998) and dimerization of the α-factor receptor (a yeast GPCR) occurs with equivalent efficiency in the ER and in the plasma membrane (Overton and Blumer, 2002). The second aspect to be considered is the assessment of the interfaces for RRIs and the intermolecular forces involved. Various models have been suggested that are not necessarily mutually exclusive, as RRIs can involve different interfaces according to (1) the type of GPCR involved; (2) the site where the interaction occurs, the ER, the GA, or the plasma membrane; and (3) whether the RRI is constitutive or triggered by chaperone molecules or, at membrane level, by scaffolding proteins or ligand binding. The contribution of all these factors has not been thoroughly investigated yet; to date, more direct and simple approaches have been carried out. Through a careful examination of various structures of adrenergic receptors by means of molecular dynamics, Gouldson et al. (2000) suggested that the role of the Journal of Molecular Neuroscience

Agnati et al. agonist might be that of stabilizing the 5–6 dimer through conformational changes in helices 5 and 6. It is important to emphasize that functional sites have been identified on the external face of helix 2, which could be involved in the formation of heterodimers; in the formation of high-order hetero-oligomers; as well as in heterodimerization processes with other proteins such as receptor activity-modifying proteins (RAMPs) and ion channels. Our group has developed this model further by observing that even considering only contact 5–6 dimers and swapped 5–6 dimers, three basic models can occur: (1) pure swapping; (2) pure contact; and (3) mixed model (contact and swapping). By taking advantage of both contact and swapping, the formation of high-order oligomers can take place. A possible mechanism underlying the formation of high-order oligomers based on the mixed model is shown in Fig. 8 (Agnati et al., 2004a). It should be noticed how simple the formation of both linear oligomers (see Fig. 8) and close-loop oligomers can be. It is sufficient that the free helices 6 of the first GPCR take contact with the free helices 5 of the last GPCR (contact dimerization). It is obvious that helices 2 could also be involved in the formation of highorder oligomers, as well as the amino and carboxyl termini of the GPCR, as was postulated previously (Agnati et al., 1995). Asimilar linear model has been suggested recently by Lee et al., based on data demonstrating that in family 1 GPCRs, a transmembrane (TM) domain interaction is involved and, in particular, TM domain 4 (TM4) has been identified as a symmetrical interface in D2 receptor (D2R) dimerization (Guo et al., 2003; Lee et al., 2003b). Lee based his linear model on the assumption that there is both a symmetrical dimer interface involving TM4 and an additional interaction involving helix 8 (a nonmembrane spanning helix between TM7 and the carboxyl terminus) with the third intracellular loop (Lee et al., 2003a). According to this model, a chain of receptor dimers can be formed congruous with the atomic force microscopy images of oligomeric rhodopsin organized into rows of dimers (Fotiadis et al., 2003). The analysis of the intermolecular forces between monomers to form dimers and between dimers to give rise to high-order oligomers is of basic importance to clarify the plasticity/stability of the macromolecular complex. Data are available demonstrating that the intermolecular forces between receptor dimers within higher-order oligomers are disrupted more readily than the intermolecular forces between monomers

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Fig. 8. Schematic representation of a possible linear model of oligomer formation. This model is based on an extension of the proposal of Gouldson et al. (2000). The scheme is a slight modification of a previously published figure (Agnati et al., 2002). Also the proposal of Lee’ et al. (2003a) of dimers as building blocks for the high-order oligomers has been considered. The C and N-terminus can be involved in the formation of high-order oligomers (see, for example, Kammerer et al. 1999) (for further details, see text).

forming dimers. However, the forces involved in GPCR oligomerization are probably different according to the GPCR considered; and these forces act not only between two interfaces of the receptors but, conversely, multiple interfaces play a role (Lee et al., 2003a). Data are available that indicate low-energy requirements for receptor–receptor contact, whereas highenergy levels are required for receptor–receptor swapping. Gouldson and colleagues (2000) have evaluated, by means of molecular dynamics simulations, the energy of different models of dimer formation for adrenergic receptors. It has been shown that the 5–6 domain-swapped dimer is a high-energy structure both in the absence of ligands and in the presence of antagonist. However, in the presence of an agonist, the energy of the 5–6 domain-swapped dimer is a lowenergy structure. Thus, the 5–6 domain-swapped dimer might be the active (high-affinity, R*) form of the receptor that interacts with the G protein. Furthermore, the agonist in some GPCRs might stabilize the 5–6 domain-swapped dimer through conformational changes in helices 5 and 6.


According to Lee’s data, oligomerization of the receptors occurred prior to cell-surface trafficking and a properly arranged oligomeric complex was required for D2R trafficking (Lee et al., 2003a). The important observation that intermolecular interactions between monomers are more stable than intermolecular interactions between receptor dimers supports the view that receptor dimers represent the building blocks of high-order oligomers (Lee et al., 2003a). For family 3 GPCRs, other domains have been shown to be involved. In the case of GABAB receptors, a coiled-coil interaction between C-terminal domains is involved (Pagano et al., 2001); in the case of metabotropic glutamate receptors, disulfide bridges in the extracellular part are involved (Robbins et al., 1999). Furthermore, as pointed out by Parmentier et al. (2002), for family 3 GPCRs, the ligand-binding (B) domain is clearly distinct from the heptahelical effector (E) domain. These investigators postulate that B and E domains are functionally coupled in such a way that there is a place for the occurrence of other subtle modulations

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148 between the binding and the transducing action. The B domain is made by two lobes that can be either in an open (Bo) or in a closed (Bc) conformation. In the presence of an agonist, the ligand is trapped in the Bc conformation. The E domain can also oscillate between active (E*) and inactive (E) conformations. It is believed that the functional coupling between B and E depends on a constant (β > 1). Thus, the Bc conformation favors the E* conformation; and conversely, the E conformation can affect the B conformation. According to our discussion, it can be hypothesized that this family of receptors is under several modulatory influences, such as the following: 1. 2. 3. 4.

5.

Effects of ion activities (e.g., protons) in the receptor microenvironment on the conformation of the B domain. Interactions of the B domain with other extracellular loops of GPCRs. Interactions of the E domain with intracellular signals and/or phosphorylation/desphorylation processes of this domain. Interactions with other receptors can affect the β constant. A RRI can directly alter the modulation of the allosteric interaction between the B and E domains; hence, the efficacy of the reciprocal control between these two functional domains of the receptor occurs. However, the RRIs might also alter the β constant indirectly by modulation of the K1 and/or K2 constants controlling the equilibrium between the closed/open state of the binding pocket and between the active/inactive state of the effector, respectively. Interactions with other receptors can affect the B and E domains, leading to some sort of independence for the state of one domain with respect to the state of the other domain without affecting the β constant.

All of these first-hand modulations can be arranged in different temporal sequences leading to complex regulatory processes and, hence, to special information handling by the receptor that can work as a microcomputing device (Agnati et al., 2002). A GPCR can therefore have different functional properties according to its state of monomer, homooligomer, or hetero-oligomer and also according to the type of interactions that links the receptor to the other receptors in the mosaic. The type of RRIs that a receptor under scrutiny can perform is an important feature that should be considered for classification of GPCRs (Wess, 1998; Lefkowitz, 2000). For example, an important criterion for GPCR classification could be the assessment of whether the dimerization process (or, more generally, a certain protein–protein interaction) is a


Agnati et al. necessary step in receptor activation (conclusive experimental evidence is presently available only for GABAB receptors). There is a very clear example of allosteric interactions between dimer partners in the case of GABAB, where it has been shown that agonist binding occurs only at the GABAB-R1 N-terminal-binding domain, and the G-protein coupling has been mapped to the GABAB-R2 intracellular domain (Robbins et al., 2001). Furthermore, it has been shown that G protein activation might require a dimeric GPCR interacting with a single heterotrimeric G protein (Breitwieser, 2004). Thus, it could also be suggested that the G protein heterotrimer can, in some instances, operate as a scaffolding protein to at least stabilize a dimer. However, our view suggests that several possible interactions occur also between the same partners according to the conditions in which the interaction takes place; therefore, an array of possible different oligomers can be formed (high plasticity of RMs). It can be surmised that in some cases there are oligomers of stably interacting receptors forming the backbone of the RM (strong intermolecular forces are in operation), and on this backbone satellite receptors (weak intermolecular forces are in operation) contribute to the formation of highly plastic RM, which can carry out several different integrations of extra- and intracellular signals because of its overall structure and adaptability. That is, the RM (composed by the backbone structure and the satellite molecules) can be modified very easily by maintaining the backbone and changing the satellite receptors (and/or other modifier molecules) to fulfill a new task. In this framework the possible role of weak electrostatic interactions between receptor monomers and/or dimers also should be considered. The D2R charge has a positive charge in the intracellular part of its heptahelical molecule. As pointed out above, this charge could attract a counter-ion atmosphere that could operate as a loose link to tether D2Rs, allowing some weak interactions that might have a role for a more stable D2R dimer assembly. Thus, as far as the functional aspects are concerned, we have reached a crucial point. It has been determined that the function of a receptor depends on its active state (constitutive or ligand induced) and on the activation of an effector. These events will, inter alia, depend on (1) where it is located in a horizontal network, in particular if it belongs to a certain RM; (2) how close the GPCR is to the effector in the

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molecular network; and (3) the type of protein– protein interactions in which the receptor takes part. These parameters can affect both the acquisition of the active state by the receptor and the effector to be activated. Because RMs are connected with effector proteins (e.g., enzymes and ion channels) and work as an integrative unit in HMNs, it has been proposed that RMs at the synaptic level can affect the efficacy of the synaptic transmission (synaptic weight) and therefore might have a role in learning and memory processes (see below; Agnati et al., 2002, 2003b). This was the basic idea that led us to introduce the RM hypothesis of the engram (Agnati et al., 1982).

dimers to gate the channel directly. Increase in channel activity can also be mediated by activation of protein kinase A (PKA), following the activation of GPCRs linked to Gαs-type G proteins. Thus, when this control mechanism of GIRK occurs within caveolae (in the special LRs enriched with these proteins), caveolins can regulate GIRK activity. These data should be interpreted in light of the demonstration that D2Rs form a stable complex with GIRK/Kir3 channels and that Gβγ is critical for D2R/Kir3 complex formation but not for maintenance of the complex (Lavine et al., 2002).

Ion Channels as a Special Class of Outputs (Effectors) of HMNs Some aspects of the structural and functional relationships between HMN inputs and HMN effectors are discussed here. Our analysis is focused on GPCRs (as a special class of inputs to HMNs) and ion channels (as a special class of outputs of HMNs). Of interest in the present analysis of input/effector relationships in the HMNs are the G protein-coupled inward-rectifying potassium channels (GIRK/Kir3) that play an important role in controlling cellular excitability. Lipid rafts (LRs) might be detected by immunocytochemistry against GM1 labeled with the cholera toxin B-subunit (Harder et al., 1998). Lipid rafts (LRs) show a structural polarization as, for example, membrane proteins anchored by a glycosyl/phosphatidyl /inositol link accumulate on the extracellular face of the LRs, whereas G proteins and members of the Src family of protein tyrosine kinase are found associated with the inner leaflet of the LRs (Paratcha and Ibanez, 2002). It seems possible also that phosphatidylinostiol 4,5-biphosphate (PIP2) and its metabolites (phosphatidylinostiol 3,4,5-triphosphate [PIR3] and DAG) are preferentially localized to GM1 patches. A spatial coupling between the outer leaflet raft lipid (GM1) and inner leaflet lipids (PIP2, PIP3, DAG) has been demonstrated in T-cells (Parmryd et al., 2003). It is interesting to note that PIP2 is closely associated with the channel GIRK to stabilize its functional integrity. Reduction of GIRK activity can be mediated by GCPRs that are associated with Gq-type G proteins activating PLC, which breaks down PIP2 (Sadja et al., 2003). On the contrary, increase of the channel activity of GIRK can be mediated by the activation of GPCRs associated with Gi/o-type G proteins, which release Gβγ

It seems clear that the plasma membrane is not a uniform bilayer but, conversely, contains islands of markedly different chemical composition and structural characteristics (Agnati et al., 1990; Gil et al., 1998; Paratcha and Ibanez, 2002); although the final evidence is still missing (Munro 2003). It has been assessed that a high diversity of membrane lipid species is present at the membrane level (between 500 and 1000), and these findings give an idea of the possible high diversity of microdomains at the plasma membrane level (Maxfield, 2002; Martsens et al., 2004). Actually, it has been suggested that similar to the ensemble of genes (the genome) and proteins (proteome), each individual has a well-preserved lipid composition (lipidome) (Sackmann and Bruisma, 2002). In agreement with this view is the evidence that lipids in the membrane do not have simply a supporting role or do not act just as a barrier between the extra- and intracellular environment but also have effects on the type of molecules that will be associated and/or inserted in the membrane, as well as having a role as informational molecules (both in the plane of the membrane, e.g., DAG, and from the plane of the membrane, e.g., PI3). Finally, endocytotic pathways have been clarified with their involvement in lipid-based mechanisms that couple membrane dynamics and protein sorting (Van der Goot and Gruenberg, 2002). Hence, it is important to understand the molecular mechanisms determining location in the membrane, geometry (x–y dimension, thickness, and shapes), chemical composition, and turnover of these microdomains. For example, is the trigger for the formation of a LR in the plasma membrane the insertion of a special class of proteins and/or lipids? How are these triggering molecules targeted to the appropriate location?


General Discussion

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150 Some features of the z dimension are presently being elucidated. Examples have shown that the coupling between leaflets is necessary for organizing inner leaflet membranes into analogous domains (Maxfield, 2002). Likely, a careful analysis of the x,y,z characteristics of LRs will demonstrate plastic changes of their geometry and chemical composition in different functional conditions. For example, it has been shown that when neutrophils polarize and begin to move in response to a chemoattractant, protein CD44 is swept to the rear of the cell by an actin/myosindependent process, and the CD44-enriched region coincides with a large area in which lipids are more resistant to extraction. This suggests that micron-scale collection of raft domains could organize in cells as they polarize. Such domains were also observed in polarized T-lymphocytes (Maxfield, 2002). All of these aspects can help in the understanding of RM formation and, more generally, the formation of HMNs. It is probable that LRs and the scaffolding, adapter, and anchoring proteins orient RRIs (and, hence, the structural and functional characteristics of the RMs) as far as (1) location of the RM in the LR or, more generally, in the plasma membrane; (2) chemistry of the RRIs and of the interactions with other proteins (e.g., effector proteins such as channels and enzymes); (3) stability of the RM and hence its transient or more long-lasting modulatory actions on cell function; and (4) sequential order according to which the information travels in the RM (Agnati et al., 2002). Another very difficult problem is what rules govern molecular interactions occurring between proteins, lipids, and carbohydrates and across these different classes of molecules. This issue has some similarity with discovering rules governing the fitting of a threedimensional assembly of a huge number of different pieces of a mosaic in which the shape and chemical characteristics of each piece play a role and each piece is plastic and changes according to the microenvironment in which it is embedded. We have briefly illustrated the importance of electrostatic interactions and the lipid microenvironment on some aspects of protein–protein interactions, but this is clearly a small light lost in the darkness. In discussing the multiple switchboards present at plasma membrane level (Okamato et al., 1998) or the multiple HMNs present in the various microdomains of the plasma membrane (Agnati et al., 2002, 2003a), it remains unclear whether these computational arrangements are preformed and thereafter inserted in the membrane or whether they are assembled from single constitutive elements at the plasma


Agnati et al. membrane level. Probably they are preassembled as building blocks (see, e.g., the case of receptor dimers) and then the HMN is finally arranged at the plasma membrane level starting from these building blocks and in sites where membrane scaffolding and adapter proteins play a major role. The formation of the HMNs also should be deeply affected by the extracellular signals that can orient the clustering of receptors into RMs and the association of the various RMs with a set of effectors (channels and enzymes). Other extracellular signals besides the classical and nonclassical neurotransmitters can affect the microdomain organization of the plasma membrane and the formation of HMNs. Among these extracellular signals, trophic factors and extracellular matrix proteins should be mentioned. It is conceivable that extracellular signals can trigger processes favoring the formation in the membrane of the proper lipid platform in which suitable proteins (maybe in a first phase, receptors for extracellular matrix protein, trophic factors, and only later on for neurotransmitters) are inserted (Lewis et al., 2001; Vereb et al., 2003). These receptors can form the first RMs and HMNs. The action of these molecular networks can then orient the further development of RMs and HMNs in that plasma membrane microdomain. Thus, the hypothesis is given that RMs can represent crucial integrative-switcher devices capable of orchestrating the structural and functional characteristics of HMNs by organizing around them the molecules taking part in the network, as well as by choosing in a flexible way the effectors to be activated (inhibited) in response to the extraand intracellular signals. Hence, RMs in HMNs can, for example, select the type of G proteins to be activated/inhibited (likely more than one at the same time) and the type of ion channels to be gated or nongated (likely more than one at the same time). To test this hypothesis it will be of paramount importance to characterize RMs in the HMNs and to understand their modes of operation. To this aim, various RRIs are being studied in relation to the structural and functional features of LRs.

Acknowledgments This work has been supported by a grant from the European Commission (QLG3-CT2001-01056). We thank Professor Giorgio Baccarani for reviewing the manuscript.

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Suggested Reading Park P. S.-H., Filipek S., Wells J.W., and Palczewski K. (2004) Oligomerization of G protein-coupled receptors: past, present, and future. Biochemistry 43, 15,643–15,656.

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