Phosphoinositide Recognition Domains - Wiley Online Library

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Traffic 2003; 4: 201–213 Blackwell Munksgaard

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Blackwell Munksgaard 2003 ISSN 1398-9219

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Phosphoinositide Recognition Domains Mark A. Lemmon Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA19104, USA, [email protected] Domains or modules known to bind phosphoinositides have increased dramatically in number over the past few years, and are found in proteins involved in intracellular trafficking, cellular signaling, and cytoskeletal remodeling. Analysis of lipid binding by these domains and its structural basis has provided significant insight into the mechanism of membrane recruitment by the different cellular phosphoinositides. Domains that target only the rare (3-phosphorylated) phosphoinositides must bind with very high affinity, and with exquisite specificity. This is achieved solely by headgroup interactions in the case of certain pleckstrin homology (PH) domains [which bind PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2], but requires an additional membrane-insertion and/or oligomerization component in the case of the PtdIns(3)P-targeting phox homology (PX) and FYVE domains. Domains that target PtdIns(4,5)P2, which is more abundant by some 25-fold, do not require the same stringent affinity and specificity characteristics, and tend to be more diverse in structure. The mode of phosphoinositide binding by different domains also appears to reflect their distinct functions. For example, pleckstrin homology domains that serve as simple targeting domains recognize only phosphoinositide headgroups. By contrast, certain other domains, notably the epsin ENTH domain, appear to promote bilayer curvature by inserting into the membrane upon binding. Key words: endosome, FYVE, headgroup, membrane, PH, phox, pleckstrin, PX, targeting Received 12 December 2002, revised and accepted for publication 8 January 2003

Controlled relocalization of proteins to specific membranes at particular times is critical for the regulation of many intracellular signaling and trafficking events. For example, signaling from cell-surface receptors requires membrane recruitment of phospholipase C (PLC), phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) isoforms. Similarly, endocytosis and trafficking from one intracellular compartment to another often require multiple proteins to be recruited to a membrane surface, but only when a relevant cargo is present in a particular lipid

context. Localization of many such proteins to membrane surfaces is driven by the reversible association of one or more domain(s) within the protein with membrane lipids (1). Two primary mechanisms have been described for the control of lipid–mediated membrane association by these ‘conditional’ peripheral membrane proteins:

1. The domain may bind to an abundant membrane lipid, but its affinity for that lipid may be modulated. This mechanism is employed by C2 domains (for PKC homology-2), for example, which bind abundant phospholipids such as phosphatidylserine. Calcium binding dramatically alters the electrostatic properties of many C2 domains, and promotes their association with these lipids (2,3). 2. The domain may recognize a membrane lipid that is present in the membrane only at specific locations, or only in response to particular cellular stimuli. For example, certain C1 domains bind diacylglycerol (2), produced when PLCs are activated. Several other types of domain specifically recognize phosphoinositides, which can be restricted to particular membranes, at concentrations that can be acutely regulated (4). In these cases, appearance of the target lipid in the membrane is directly responsible for membrane recruitment of the interacting protein.

Membrane targeting by C1 and C2 domains has been excellently reviewed elsewhere (1,2), so I shall focus in this article on domains that interact primarily with phosphoinositides. Phosphoinositides are phosphorylated forms of phosphatidylinositol (PtdIns), present at relatively low levels within the cell. They are unique among phospholipids in their ability to be modified rapidly by headgroup phosphorylation/dephosphorylation, to transiently create (or eliminate) membrane-targeting signals at particular locations. Domains shown to recognize phosphoinositides now include FYVE, PX (phox homology), PH (pleckstrin homology), ENTH, ANTH, Tubby, and FERM domains (4), as well as several other proteins involved in the control of endocytosis and/or cytoskeletal assembly (Table 1). There is significant diversity at both a structural and a functional level in domains that bind phosphoinositides, which I shall attempt to illustrate in this article. 201

Lemmon Table 1: A list of phosphoinositides and domains that recognize them Phosphoinositide

Target domains (in vivo)

PtdIns(3)P PtdIns(4)P PtdIns(5)P PtdIns(3,4)P2 PtdIns(3,5)P2 PtdIns(4,5)P2

FYVE, PX (PH?) PH (78) none identified PH (PX?) none identified PH, FERM, ANTH, ENTH, tubby (PX?), AP2-a, plus several cytoskeletal proteins PH

PtdIns(3,4,5)P3

Phosphoinositides and Phosphoinositide Recognition The phosphoinositide cycle in mammalian cells is summarized in Figure 1. No clear binding partners have yet been reported for either PtdIns(5)P or PtdIns(3,5)P2, first detected in 1997 (5,6). However the remaining 5 different phosphoinositides have all been shown to contribute to membrane targeting of one or other conditional peripheral membrane protein in yeast or mammalian cells. Precisely how each phosphoinositide contributes to membrane

targeting, however, appears to depend on its structure and cellular abundance (7). In particular, while each of the rare phosphoinositides (those with a phosphate group at the 3-position) appears to recruit only a small subset of domain types (presumably those best suited to recognizing that particular lipid), the more abundant phosphoinositide PtdIns(4,5)P2, which accounts for approximately 1% of lipid molecules in the plasma membrane, recruits a wider array of different membrane-association domains. To illustrate this point, it is most instructive to consider the phosphoinositide-recognition domains classified by phosphoinositide species:

PtdIns(3,4,5)P3: A PH Domain Target in Intracellular Signaling PtdIns(3,4,5)P3 is the major product of agonist-stimulated PI 3-kinases (7). It is present at very low levels in unstimulated cells, but its concentration increases by more than 40-fold within seconds of stimulation. A domain capable of specific PtdIns(3,4,5)P3 recognition, but which does not bind significantly to other phosphoinositides, can be recruited rapidly and transiently to the plasma membrane of agonist-treated cells in a PI 3-kinase-dependent manner. The only domains known to exhibit these PtdIns(3,4,5)P3-binding properties

Figure 1: Relationships between phosphoinositides found in mammalian cells. PtdIns, the most abundant lipid in this scheme, can be phosphorylated at the 3-, 4-, and 5-positions. PtdIns(4)P accounts for approximately 95% of the PtdInsP, and PtdIns(4,5)P2 accounts for more than 99% of the total PtdInsP2. PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are found in mammalian cells but not yeast cells, and are only found after stimulation with growth factors or other agonists (7). Gray arrows represent less well-characterized pathways, or pathways through which a minority of the phosphoinositide is generated.

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are pleckstrin homology (PH) domains from such molecules as protein kinase B (PKB), Bruton’s tyrosine kinase (Btk), the general receptor for phosphoinositides-1 (Grp1) (8–10), and the dual adaptor for phosphotyrosine and 3-phosphoinositides-1 (DAPP1) (10–12). In each of these cases, rapid and dramatic PI 3-kinase-dependent plasma membrane recruitment of a green fluorescent protein (GFP)/PH domain fusion protein can be visualized directly, following stimulation of cells with many different agonists (1). PH domains are structurally well-characterized modules of approximately 120 amino acids (9). Those that specifically recognize PtdIns(3,4,5)P3 represent only a very small minority – perhaps 5–10% – of all PH domains in the human proteome (10). All PH domains share a common core fold that consists of a 7-stranded b-sandwich formed from two near-orthogonal b-sheets (Figure 2). Because of the twist in the antiparallel b-sheets, two corners of the b-sandwich are ‘splayed’ open (top and bottom in Figure 2), while the other two are ‘closed’ (left and right in Figure 2). One splayed corner is covered by the characteristic C-terminal

a-helix of PH domains (top in Figure 2), and the other is covered by three interstrand loops that form much of the phosphoinositide-binding site (bottom in Figure 2). PH domains are also electrostatically polarized, with their positively charged face coinciding with the phosphoinositidebinding site (9). The 20 or so PH domains that specifically recognize PtdIns(3,4,5)P3 (of some 250 PH domains in the human proteome) all contain a sequence motif defined by a pattern of basic residues centered on the loop between the first two b-strands (10). Crystal structures of PH domains with this motif, bound to the PtdIns(3,4,5)P3 headgroup, showed that the motif allows a spatial disposition of basic side-chains that complements the arrangement of phosphate groups in the headgroup to maximize hydrogenbonding interactions (13–16). The isolated PtdIns(3,4,5)P3 headgroup appears to bind at least as strongly to these PH domains as does the intact membrane-embedded phosphoinositide (12), arguing that headgroup interactions alone can drive membrane recruitment. The headgroup binds within a well-defined binding pocket, and multiple hydrogen bonds are made to each available phosphate group. A total of 17–19 hydrogen bonds are made between PtdIns(3,4,5)P3-specific PH domains and the bound headgroup (14). Specific PH domain/PtdIns(3,4,5)P3 complexes, especially those of the Grp1 and Btk PH domains (17,18), represent the strongest univalent phosphoinositide interactions known.

PtdIns(3,4)P2: A Lipid Second-Messenger Target of Some PH Domains, and a PX Domain

Figure 2: A ribbons representation of the Grp1 PH domain bound to the PtdIns(3,4,5)P3 headgroup [Ins(1,3,4,5)P4]. The 7 b-strands that form the core PH domain b-sandwich are labeled b1 through b7, and the characteristic C-terminal a-helix of PH domains is labeled a1. Grp1-PH has two additional b-strands (b60 and b600 ) that contribute to headgroup recognition. Phosphate groups in the bound Ins(1,3,4,5)P4 are labeled P1 through P5. The structure was taken from Ferguson et al. (14).

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Most PtdIns(3,4)P2 in mammalian cells is thought to be generated by 5-dephosphorylation of PtdIns(3,4,5)P3 (7), although there have been reports of PtdIns(3,4,5)P3independent routes for its synthesis (19,20). In either case, PtdIns(3,4)P2 occurs at significant levels in the plasma membrane only transiently after stimulation and, like PtdIns(3,4,5)P3, is a lipid second messenger. PtdIns(3,4)P2 has several well-known targets, which are all PH domains that contain the sequence motif mentioned above. Several of the PH domains that are recruited by PtdIns(3,4,5)P3 also recognize PtdIns(3,4)P2. These include the PH domains from PKB (16) and DAPP1 (14), but not those from Btk or Grp1 [which are PtdIns(3,4,5)P3-specific] (12). PtdIns(3,4)P2 recognition by PH domains is very similar to that seen for PtdIns(3,4,5)P3 binding in Figure 2. It involves the same array of basic residues in the b1/b2 loop of the PH domain, which form a well-defined binding site that makes multiple hydrogen bonds with the phosphates in the headgroup. The difference between a PH domain that recognizes both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 and one that recognizes only PtdIns(3,4,5)P3 is quite 203

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small and can be rationalized (and indeed predicted) based on recent structural studies (14–16,21). One PH domain, from TAPP1 (tandem PH domain containing protein 1), has been reported to bind selectively to PtdIns(3,4)P2 (22), and evidence has been presented to suggest that PtdIns(3,4)P2 is the target of this PH domain in vivo (23). However, our own in vitro studies show that the C-terminal TAPP1 PH domain (from AA054961) binds to PtdIns(3,4,5)P3 only about 4-fold more weakly than it does to PtdIns(3,4)P2 (14). It is therefore not clear that this is a bona fide PtdIns(3,4)P2-specific PH domain, although it does represent the only known example for which PtdIns(3,4)P2 is the highest-affinity ligand. Recent studies have also suggested that a single phox homology, or PX, domain (from p47phox) may bind selectively to PtdIns(3,4)P2 (24,25) [although, as discussed below, most PX domains prefer PtdIns(3)P].

PtdIns(3)P: A Key Phosphoinositide in Membrane Trafficking, Recognized by FYVE and PX Domains PtdIns(3)P is produced by the class III PI 3-kinase Vps34. In mammalian cells, PtdIns(3)P is found primarily in membranes of early endosomes and the internal vesicles of multivesicular endosomes (26). It is also found on vacuolar and endosomal membranes in yeast (26). A large body of work has shown that PtdIns(3)P plays important roles in regulating trafficking in the early endocytic pathway (27) as well as in Golgi to lysosome/ vacuole sorting (28), and appears to serve as a ‘marker’ for endosomal membranes. Of all physiological phosphoinositides, PtdIns(3)P is the one that has the largest number of known specific binding partners, being recognized specifically by most FYVE domains and PX domains, of which there are 30 and 42, respectively, in the human proteome (29). Detailed structural and biophysical studies of FYVE and PX domains have provided an excellent basis for understanding how they recognize PtdIns(3)P in cellular membranes (30–32).

FYVE domains The FYVE domain contains approximately 60–70 amino acids, and is named for the four proteins in which it was first identified: Fab1p, YOTB, Vac1p, and EEA1 (33). FYVE domains contain two b-hairpins plus a small C-terminal a-helix that are held together by two Zn2þ-binding clusters (Figure 3A) in a zinc finger-like structure (34). A basic motif in the first b-strand (R/K)(R/K)HHCR, contributes to a shallow positively charged pocket that dominates the surface of the FYVE domain and is critical for PtdIns(3)P binding. Side-chains from this motif account for nearly all hydrogen bonds seen between the PtdIns(3)P headgroup and the 204

EEA1 FYVE domain [crystallized in complex with Ins(1,3)P2] (31). In addition to those involving the 1- and 3-phosphates, hydrogen bonds are also made with the 4-, 5-, and 6-hydroxyls of the inositol chair, and it is clear from the structure that a 4- or 5-phosphate would disrupt hydroxyl-group hydrogen bonding in addition to creating steric problems. Similarly, binding of PtdIns(5)P could only be achieved by sacrificing at least one hydroxyl-group hydrogen bond. This structure has thus provided a satisfying explanation for how the FYVE domain selects its PtdIns(3)P target. In addition to providing explanations for phosphoinositide-binding specificity, structural studies of EEA1 FYVE help to explain the puzzle of additional requirements for targeting FYVE domains to membranes in vivo. Since their initial description (35), it has been clear that more than just the FYVE domain is required for efficient targeting of proteins to PtdIns(3)P-containing membranes in vivo. The reason for this is simply that PtdIns(3)P is not very abundant, and that headgroup binding, while specific, is relatively weak. Compared with 19 headgroup hydrogen bonds in a high-affinity PH domain complex, the occurrence of just 9 in the Ins(1,3)P2/EEA1 FYVE complex (31) argues that it must be a significantly weaker interaction. Indeed, the Grp1 PH domain (with its 19 hydrogen bonds) binds the PtdIns(3,4,5)P3 headgroup [Ins(1,3,4,5)P4] with a KD of 27 nM (12), while EEA1 FYVE binds the PtdIns(3)P headgroup [Ins(1,3)P2] 1000 times more weakly, with a KD of 24 mM. An interaction of this strength is certainly insufficient to explain targeting of the FYVE domain to endosomal membranes. Studies of FYVE domain binding to short-chain phosphoinositides do not support the hypothesis that there are significant interactions with the glycerol backbone or other moieties. However, it has been shown for the Hrs1 FYVE domain that binding to membrane-embedded PtdIns(3)P is preferred dramatically over binding to the isolated phosphoinositide or headgroup (36). This preference appears to result from membrane penetration by the FYVE domain, which inserts nonpolar side-chains from the membrane interaction loop (Figure 3A) into the membrane interior (37,38), as initially proposed by Misra and Hurley (34). These weak and nonspecific hydrophobic interactions will cooperate with the weak and specific headgroup interactions to aid membrane targeting of the FYVE domain. However, even the combination of headgroup association and membrane penetration does not appear to be enough for efficient membrane targeting of most FYVE domains. For example, Stenmark and colleagues could not see strong endosomal localization of the isolated Hrs1 or EEA1 FYVE domains (26,35), unless they promoted dimerization of the domain by expressing a pair of Hrs1 FYVE domains in tandem (26), or by including an N-terminal extension that promotes dimerization (for EEA1). FYVE domain dimerization increases the avidity of binding to membraneembedded PtdIns(3)P, and this is required for in vivo Traffic 2003; 4: 201–213

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Figure 3: Structures of (A) the EEA1 FYVE domain dimer (PDB code 1JOC) (31) and (B) the p40phox PX domain (PDB code 1H6H) (30) bound to Ins(1,3)P2 and dibutanoyl-PtdIns(3)P, respectively. The two domains are oriented such that the membrane in which the bound phosphoinositide would be embedded is perpendicular to the page, passing beneath the representation of the two domains. The 1-and 3-phosphates are labeled P1 and P3, respectively. Dimerization of the FYVE domain is driven by a 60 amino-acid N-terminal extension that forms a coiled-coil. In the FYVE domain, the two bound Zn2þ ions per protomer are colored gray. In the PX domain, the Y94 side-chain, which makes van der Waal’s contact with the glycerol backbone is shown and labeled (see text). The loop labeled ‘membrane interaction loop’ is thought to insert into the membrane in both cases, based on NMR chemical shift changes seen upon binding of each domain to PtdIns(3)P-containing micelles (37,42).

membrane targeting of most FYVE domains. The EEA1 FYVE structure of Dumas et al. (31) included the N-terminal coiled-coil that promotes its dimerization, and shows how the FYVE domains are likely to be arranged for multivalent, high-avidity, endosomal targeting (Figure 3A). The SARA (39) and FENS1 (40) FYVE domains are exceptions in that GFP fusions of the minimal FYVE domain are targeted efficiently to PtdIns(3)P-containing membranes in vivo. This may reflect an inherent capacity of these FYVE domains to oligomerize without additional sequences, or may suggest that they make more extensive headgroup interactions.

PX domains PX domains were pointed out in 1996 as a 130-amino acid homology region in two components of the phagocyte NADPH oxidase (phox) complex (p40phox and p47phox) as well as many other proteins with diverse functions (41). Most PX domain-containing proteins are involved in vesicular trafficking, protein sorting, or lipid modification. A flurry of papers in the summer of 2001 identified PX domains as PtdIns(3)P binding modules (24,42–46), although two examples (the PX domains from p47phox and C2 domain PI 3-kinase) have been reported to bind Traffic 2003; 4: 201–213

other phosphoinositides (24,44). In Saccharomyces cerevisiae, all PX domains identifiable in the genome bind preferentially to PtdIns(3)P (46). A large proportion of PX domain-containing proteins are sorting nexins (or SNX proteins) that have been implicated in the endocytic pathway, thus identifying a class of PtdIns(3)P effectors likely to play key role in regulating endosomal trafficking and sorting. Bravo et al. determined the crystal structure of the p40phox PX domain bound to dibutanoyl-PtdIns(3)P (30). As shown in Figure 3B, the 120 amino-acid domain has an N-terminal 3-stranded b-sheet that abuts a helical subdomain. The PtdIns(3)P headgroup binds in a pocket between the b-sheet and the helical subdomain. A total of 9 hydrogen bonds are made between the phosphoinositide and PX domain: three involving the 3-phosphate, and two involving the 1-phosphate (30). As with FYVE domains, specificity for PtdIns(3)P appears to be defined by the fact that additional phosphates would disrupt hydrogen bonds formed by the 4- and 5-hydroxyl groups, and could not be accommodated without significant steric clashes. Also in common with FYVE domains, the relatively small number of hydrogen bonds between the PX domain and PtdIns(3)P argues that isolated Ins(1,3)P2 is likely to bind with only low affinity. In the complex between p40phox-PX and 205

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dibutanoyl-PtdIns(3)P, a tyrosine side-chain (Y94) appears to make additional van der Waal’s contact with the glycerol backbone of the phosphoinositide. This tyrosine is present within the ‘membrane interaction loop’ (Figure 3B), in which substantial NMR chemical shift differences are seen upon PX domain binding to PtdIns(3)P-containing micelles (42). As with FYVE domains, it therefore appears that penetration of the bilayer may play an important role in increasing the affinity of PX domain binding to PtdIns(3)P-containing membranes. Several PX domains (including that from p40phox) are efficiently targeted to PtdIns(3)P-containing membranes when expressed in isolation (24,43,45). This targeting does not require PX domain oligomerization (30), arguing that the combination of relatively weak headgroup binding and bilayer insertion is sufficient for high-affinity binding. However, this is not true for all PX domains. In a survey of S. cerevisiae PX domains, we recently found that while four (of 15) PX domains bind strongly to PtdIns(3)P-containing membranes in vitro, and are likely to be capable of independently driving endosomal localization in vivo (46), the remaining 11 PX domains bind only very weakly (yet appear to retain their PtdIns(3)P specificity). The majority of the side-chains involved in PtdIns(3)P headgroup interaction (30) are conserved in most PX domains, regardless of whether they bind strongly or weakly to PtdIns(3)P, allowing for the possibility that headgroup recognition is broadly similar in all cases. PX domains differ significantly, though, in the sequence and length of their membrane interaction loop, where several PtdIns(3)P contacts are made in the p40phox structure (30), and where significant changes are seen upon PtdIns(3)P binding to the Vam7p PX domain (42) (a high-affinity PX domain). The sequences in this region are too variable for any precise comparative statements to be made. However, we suggest that highaffinity PX domains may have an ‘effective’ membrane interaction loop that can raise the affinity of the monomeric PX domain for PtdIns(3)P-containing membranes, while the low-affinity PX domains may have an ‘ineffective’ membrane interaction loop that cannot interact efficiently with the glycerol backbone of PtdIns(3)P and/or fails to penetrate the membrane. In yeast, it is notable that many of the proteins with low-affinity PX domains are involved in multiprotein complexes. For example, most sorting nexins have coiledcoil regions that drive their homo- and/or hetero-oligomerization, which is critical for endosomal targeting (47,48). In yeast, the sorting nexin orthologues that contain such coiled-coil sequences all have PX domains that fall into the low-affinity category. By contrast, the two yeast SNX orthologues that have high-affinity PX domains [SNX3/Grd19p and Yhr105wp (46)] both lack potential oligomerization sequences [as does mammalian SNX3 (45)]. It therefore seems reasonable to propose that PX domains required to target monomeric proteins to endosomal locations are in 206

the high-affinity class, while those that target only protein assemblies or complexes are in the low-affinity class. Mechanistically, how such low-affinity PX domains might operate is illustrated by considering the ‘retromer’ complex in yeast (49,50), which forms a membrane coat required for cargo retrieval from endosomes to the late Golgi (51). The sorting nexin orthologues Vps5p and Vps17p both contain low-affinity, PtdIns(3)P-specific, PX domains and are components of the retromer complex. A Vps35p/Vps29p/ Vps26p subcomplex appears to bind to the cargo proteins destined for Golgi retrieval. Simultaneous interaction of Vps5p and Vps17p to this complex and (via their PX domains) multivalently to PtdIns(3)P in the endosomal membrane is thought to restrict retromer complex assembly to membranes that contain both the relevant cargo and PtdIns(3)P (49). Such ‘coincidence-detection’ would not be possible if Vps5p and Vps17p contained high-affinity PX domains.

Contrasting Phosphoinositide Binding by PH Domains With That Seen for FYVE and PX Domains Phosphoinositide-specific PH domains such as those from Grp1 and Btk can achieve a sufficiently high affinity and specificity for their multiply phosphorylated targets by binding headgroup alone, while FYVE and PX domains cannot. This may simply reflect the fact that the PtdIns(3)P headgroup cannot make as many hydrogen bonds and electrostatic interactions as can the headgroups of PtdIns(3,4,5)P3 or PtdIns(3,4)P2, because it does not have as many phosphates. To circumvent this problem, FYVE domains and PX domains must utilize additional modes of interaction, not seen with PH domains, to achieve similarly high affinity interactions [levels of PtdIns(3)P and PtdIns(3,4,5)P3 are similar in activated cells (7)]. These additional interactions amount, for the most part, to inserting a loop bearing hydrophobic sidechains into the apolar interior of the membrane. Thus it is no accident that high-affinity (or reasonably high-affinity) PtdIns(3)P-targeting modules share several characteristics with one another, yet are significantly different in nature from PH domains. By analogy with this argument, one might expect that high-affinity PtdIns(5)P- or PtdIns(4)P-targeting domains will be more like FYVE or PX domains than like PH domains. PtdIns(3,5)P2 and PtdIns(4,5)P2, on the other hand, might be expected to make sufficiently strong headgroup-only interactions with PH domains [as is seen with the PLC-d1 PH domain and PtdIns 4,5)P2 (52)]. Although only FYVE and PX domains have been identified as physiologically relevant PtdIns(3)P targets, a few PH domains have been reported to select this phosphoinositide (with low affinity) in vitro (22,53). These isolated PH domains are not known to be targeted to endosomal Traffic 2003; 4: 201–213

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membranes when studied in isolation, and it is not clear whether their host proteins associate with PtdIns(3)P-containing membranes. Nonetheless, the possibility should be considered that these PH domains might resemble low-affinity PX domains in their relationship with PtdIns(3)P.

PtdIns(4,5)P2-binding domains in having a primary requirement to bind Ins(1,4,5)P3, one of the products of PLC-d1 action. In vitro, Ins(1,4,5)P3 (i.e. product) accumulation displaces PLC-d1 from PtdIns(4,5)P2-containing membranes and acts as a noncompetitive inhibitor of the enzyme (57,58). A similar Ins(1,4,5)P3/PtdIns(4,5)P2 competition may be important for PLC-d1 regulation in vivo.

PtdIns(4,5)P2: A Multifunctional Target for Many Less Discriminating Domains

As reviewed in detail elsewhere (9), most PH domains that bind PtdIns(4,5)P2 are quite nonspecific in their phosphoinositide binding. They also tend to bind PtdIns(4,5)P2 rather weakly, and most are not independently capable of targeting their host protein to the plasma membrane. Rather, most such PH domains appear to contribute to membrane association by binding PtdIns(4,5)P2 as just one part of a multi-domain interaction. For many PH domains (probably the majority), the functional importance of phosphoinositide binding has yet to be established.

Although it only accounts for approximately 1% of lipid in the plasma membrane of a typical mammalian cell, PtdIns(4,5)P2 is the most abundant (by a factor of at least 25) of the phosphoinositides for which interaction domains are known. Consequently, PtdIns(4,5)P2-targeting domains have somewhat less stringent requirements than domains that must target 3-phosphoinositides. Domains that specifically recognize PtdIns(3,4,5)P3 or PtdIns(3)P must be able to select against PtdIns(4,5)P2 or PtdIns(4)P, which are present in the cell at levels that are at least 25-fold higher (7). By contrast, it is almost true to say that a domain that binds phosphoinositides in general will tend, in vivo, to bind predominantly to PtdIns(4,5)P2 because of its relative abundance. Furthermore, since their target lipid is much more abundant, PtdIns(4,5)P2-binding domains do not need to be capable of such high affinity interactions as with PtdIns(3,4,5)P3 binding domains, for example. There are many proteins that bind PtdIns(4,5)P2 with different degrees of specificity and affinity, and which play important roles in controlling endocytosis, exocytosis, and cytoskeletal rearrangements [reviewed in (54)]. As one might expect since the requirements for PtdIns(4,5)P2 targeting are less stringent than for less abundant phosphoinositides, there are several ways in which it can be achieved, using a variety of different protein structures. PH domains: The PLC- d1 PH domain is the exception rather than the rule Perhaps the best known PtdIns(4,5)P2-binding domain, the PH domain from PLC-d1 (PLCd-PH) (9), is probably an exception among domains that interact with this lipid, and is certainly unique among PH domains. PLCdPH, used by many (fused to GFP) as a probe for PtdIns(4,5)P2 localization in vivo (1,9), binds strongly to PtdIns(4,5)P2 through highly specific interactions with its headgroup [Ins(1,4,5)P3]. In fact, PLCd-PH binds to Ins(1,4,5)P3 almost 10-fold more strongly than it does to PtdIns(4,5)P2 (52), and does so using a pattern of basic residues similar to that utilized in ligand binding by PtdIns(3,4,5)P3-specific PH domains. Although very useful as an experimental tool (55,56), the functional importance of specific, high-affinity PtdIns(4,5)P2 binding by PLCd-PH is not clear. It may be unique among Traffic 2003; 4: 201–213

Superhelical domains that link endocytic components to PtdIns(4,5)P2 Phosphoinositides, most particularly PtdIns(4,5)P2, are well known to be important for the initial steps in receptormediated endocytosis (59). Gaidarov and Keen showed that phosphoinositide binding is important for recruiting the AP2 adaptor complex to the plasma membrane, and identified an N-terminal 80 amino-acid region of the AP2 a-subunit that is responsible (60). This interaction was recently visualized crystallographically, with the impressive determination of the entire AP2 core structure with a bound inositol phosphate (61). As expected (60), the inositol phosphate binds primarily to the N-terminus of the a-subunit, which is a curved a-helical superhelical solenoid (Figure 4A). The inositol phosphate lies on the surface of the helical domain rather than within a binding pocket, consistent with the relative promiscuity of the interaction, and is contacted only via its phosphate groups, which form hydrogen bonds with the ‘tips’ of the AP2 side-chains. Collins et al. (61) likened the interaction to ‘a ball balanced on the fingertips of a half-closed hand’. A significant number of hydrogen bonds are made to AP2-a, with 11 predicted for PtdIns(3,4,5)P3, and 8 for PtdIns(4,5)P2 (based on the InsP6-bound structure). There is some additional interaction of the inositol phosphate with the m2 subunit, which mutagenesis data argue is also important in vivo (62). The N-terminal region of AP180 and its homolog, clathrin assembly lymphoid myeloid (CALM) leukemia protein also adopt an a-helical superhelical-solenoid structure that resembles the N-terminus of AP2-a, and binds to phosphoinositides (63). This has been named the ANTH, for AP180 N-terminal homology, domain (64). The ANTH domain of CALM binds to short-chain PtdIns(4,5)P2 207

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Figure 4: Binding of phosphoinositides by a-helical superhelical solenoids. In (A), a fragment corresponding to the N-terminal 80 amino-acids of the a-subunit of AP2 is shown, with bound inositol hexakisphosphate (InsP6). This is part of the entire AP2 core complex structure (PDB code 1GW5) (61). In (B), the ANTH domain from CALM (PDB code 1HFA) (63) is shown, bound to the PtdIns(4,5)P2 headgroup [Ins(1,4,5)P3]. The inositol phosphate binds to a similar basic sequence motif on the surface of AP2-a and the ANTH domain, but differs in its orientation. Part (C) shows the ENTH domain from epsin. In the left-hand panel the unliganded ENTH domain is shown (PDB code 1EDU) (67) for comparison. In the right-hand panel, which represents the Ins(1,4,5)P3-bound structure (PDB code 1HOA) (64), an additional ordered N-terminal a-helix is seen, as marked. This amphipathic helix is thought to insert into the membrane and aid induction of membrane curvature.

(Figure 4B) through interactions that closely resemble inositol phosphate binding to AP2-a – involving a very similar sequence motif (K-X9-KKK-H/Y) in both proteins. Although the spatial arrangement of the lysine side-chains is significantly different in the two structures, the phos208

phoinositide headgroup appears to be bound ‘like a ball balanced on the fingertips’ in each case (63). The AP180 ANTH domain binds relatively weakly (KD  5 mM) to PtdIns(4,5)P2, and is quite promiscuous in its phosphoinositide interactions. However, the relative abundance Traffic 2003; 4: 201–213

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of PtdIns(4,5)P2 leaves 9little doubt that this is the likely physiological ligand. AP180/clathrin complexes appear to assemble on the plasma membrane, probably driven by multivalent ANTH domain/PtdIns(4,5)P2 interactions as clathrin mediates self-association of the complexes. The sequence-related ENTH (for epsin N-terminal homology) domain (65), which also binds PtdIns(4,5)P2, recently provided an interesting surprise (64) (Figure 4C). The epsin ENTH domain binds PtdIns(4,5)P2 in vesicles about 10 times more strongly than does the AP180 ANTH domain, and (like PLCd-PH) is independently targeted to the plasma membrane (66). The epsin ENTH domain also differs from the ANTH domain in that it can tubulate liposomes in vitro, suggesting that it may penetrate the membrane surface, and alter its curvature (64). NMR studies have shown that the PtdIns(4,5)P2 binding site in this a-helical solenoid is located quite differently from those in AP2-a or the ANTH domain (66). Most interestingly, crystallographic studies of the liganded ENTH domain showed that binding of the PtdIns(4,5)P2 headgroup leads to the ‘ordering’ of an additional amphipathic a-helix at the N-terminus (64,67). Curiously, the hydrophobic face of this additional helix is exposed on the outer surface of the protein, making it a good candidate for a region of the ENTH domain that may penetrate the membrane. This hypothesis is supported by mutagenesis studies (64), arguing that phosphoinositide binding to the ENTH domain, in addition to aiding AP2 complex recruitment, may initiate membrane invagina-

tion in endocytosis by inserting an amphipathic a-helix into the cytoplasmic leaflet and inducing membrane curvature (68). These findings with the epsin ENTH domain might give us pause to wonder whether it is a coincidence that other phosphoinositide-binding domains that penetrate membranes (i.e. endosomally located FYVE and PX domains) are also involved in a process that requires vesiculation of their target compartments.

Tubby proteins: Sequestered by PtdIns(4,5)P2 The C-terminal domain of the tubby gene product provides an intriguing model for how phosphoinositide binding can contribute to cellular signaling (69). This 260 amino-acid domain was found to bind PtdIns(4,5)P2 [as well as PtdIns(3,4)P2 and PtdIns(3,4,5)P3] sufficiently strongly that it associates constitutively with the plasma membrane. Figure 5A shows how the tubby domain binds the PtdIns(4,5)P2 headgroup. In this case, binding to PtdIns(4,5)P2 in the plasma membrane serves to sequester tubby away from its effectors (which are in the nucleus). Upon stimulation of cells with agonists for G-protein coupled receptors, PLC-b is activated, leading to PtdIns(4,5)P2 hydrolysis and the dissociation of tubby from the plasma membrane. This dissociation event could be visualized directly by monitoring GFP-tubby localization following stimulation of the M1 acetylcholine receptor (69). Tubby protein released from the plasma membrane is imported into the nucleus, where it act as a transcription factor.

Figure 5: (A) Ribbons representations of the tubby C-terminal domain and the FERM domain from radixin. (A) The tubby C-terminal domain (PDB code 1I7E) is shown bound to 1-glycerophosphoryl-inositol(4,5)bisphosphate (69). Phosphate groups are labeled P1, P4, and P5. (B) Representation of the FERM domain from radixin, bound to Ins(1,4,5)P3 (PDB code 1GE6) (77). The subdomain that closely resembles a PH domain (see text) is marked. Note that the Ins(1,4,5)P3 binds close to the C-terminal a-helix of this subdomain (in a cleft between two subdomains), rather than in the location normally seen for inositol phosphate binding to PH domains (see Figure 2).

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PtdIns(4,5)P2-binding proteins in cytoskeletal regulation A final group of PtdIns(4,5)P2-binding domains and proteins are those involved in regulating adhesion of the cytoskeleton to the plasma membrane. Measurements using optical tweezers have shown dramatically that reducing (or sequestering) available cellular PtdIns(4,5)P2 results in a large drop in cytoskeleton–plasma membrane adhesion energy (70). The origin of this effect is two-fold. Certain cytoskeletal proteins (such as spectrin, via its PH domain) are anchored to the plasma membrane through interactions with PtdIns(4,5)P2. Moreover, PtdIns(4,5)P2 levels are critical in the regulation of actin polymerization, which plays an important role in determining adhesion energy. The role of phosphoinositides in regulating the actin cytoskeleton has recently been reviewed (71,72). PtdIns(4,5)P2 binds to a polybasic region in the Wiskott–Aldrich syndrome protein (WASP). Together with Cdc42 binding, this enhances the ability of WASP to bind and activate the Arp2/3 complex, thus nucleating actin filament assembly at the membrane (73). PtdIns(4,5)P2 also associates with many actin-binding proteins, mostly by interacting with one or more short stretches of basic residues (71,72,74). In doing so, PtdIns(4,5)P2 tends to promote dynamic remodeling of the actin cytoskeleton (72). For example, PtdIns(4,5)P2 binding to profilin inhibits its ability to bind and sequester actin monomers. PtdIns(4,5)P2 also prevents actin binding by cofilin, and inhibits the ability of gelsolin to sever actin filaments and to bind actin monomers. Structural details of these PtdIns(4,5)P2 interactions have not yet been elucidated. They tend to be less headgroup specific than many other phosphoinositide-binding proteins, consistent with our expectation for proteins that interact with to relatively abundant PtdIns(4,5)P2. The current state of knowledge regarding these interactions is reviewed elsewhere (72,74). Another interesting group of phosphoinositide-binding proteins may regulate the actin cytoskeleton by modulating the availability and/or distribution of PtdIns(4,5)P2 (75,76). These proteins include GAP43 (a growth associated protein), CAP23 (a cytoskeleton-associated protein) and MARCKS (myristoylated alanine-rich C kinase substrate). Each of these proteins is myristoylated or palmitoylated, and each contains a conserved basic region. By analogy with extensive studies of MARCKS, which binds PtdIns(4,5)P2 with high affinity (but little headgroup specificity) (76), these characteristics may allow GAP43 and CAP23 to cluster PtdIns(4,5)P2 into raft-like entities. Such PtdIns(4,5)P2 clusters could effectively ‘buffer’ available PtdIns(4,5)P2 levels in the plasma membrane (76). Alternatively, they might represent primary sites to which other PtdIns(4,5)P2-binding proteins are recruited. Finally, one PtdIns(4,5)P2-mediated link between the actin cytoskeleton and phosphoinositides has been resolved 210

structurally (77), in the form of the FERM (4.1-ezrinradixin-moesin) domain from radixin, bound to the PtdIns(4,5)P2 headgroup (Figure 5B). Proteins from the ezrin-radixin-moesin (ERM) family function as cross-linkers between the actin cytoskeleton and the plasma membrane: the type of cross-linkers that, when removed, would reduce cytoskeletal adhesion energy. The C-terminal domain of the protein binds F-actin, while the N-terminal FERM domain binds both PtdIns(4,5)P2 and the cytosolic tails of integral membrane proteins. Interestingly, of the three subdomains that make up the FERM domain, one very closely resembles a PH domain (77). However, the co-crystallized inositol phosphate binds to a region quite distinct from that seen in PH domain complex structures, arguing that this is where the similarity ends. The inositol phosphate binds in a cleft between two of the FERM subdomains. A rather small number of hydrogen bonds are formed, primarily to the 4-phosphate group, consistent with the promiscuity of this interaction. It is proposed that phosphoinositide binding to the FERM domain induces conformational changes that promote simultaneous binding to the cytoplasmic tails of specific integral membrane proteins (77). A high-affinity, multivalent, interaction is thus established between the actin and plasma membrane components.

Conclusions Phosphoinositide-binding domains are diverse in structure and in how they interact with their lipid targets. At one extreme, PtdIns(3,4,5)P3-targeted PH domains interact strongly and specifically with just the headgroup of their rare phospholipid ligand. At the other extreme, short basic sequences interact electrostatically with PtdIns(4,5)P2, the most abundant of the highly charged, phosphoinositides in the inner leaflet of the plasma membrane. When combined with hydrophobic interactions afforded by acylation of such a peptide, this mode of membrane association can be very strong, as seen for the MARCKS peptide (76). Between these extremes are domains such as the FYVE and PX domains that can recognize a rare lipid headgroup specifically, but cannot bind it with very high affinity. Domains in this class augment their membrane binding capacity by inserting hydrophobic side-chains into the bilayer and/or by increasing their membrane-binding avidity through oligomerization. On a final note, it is becoming increasingly clear as our understanding grows more sophisticated that phosphoinositide-binding domains can have profound effects on the physical properties of membranes with which they interact. For example, the ENTH domain induces curvature of its target membranes (64). MARCKS and other proteins have been shown to cluster PtdIns(4,5)P2 in vitro (76). It is likely that several of the other known membrane-targeting Traffic 2003; 4: 201–213

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domains also double as ‘membrane-modulation’ domains with key roles in cellular processes.

Acknowledgments I would like to thank Kathryn Ferguson for valuable discussions and for help in making figures, and members of my laboratory for discussion and comments on the manuscript. Work in the author’s laboratory in this area is funded by NIH grant GM56846.

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