Functional role of proline and tryptophan residues highly conserved ...

The EMBO Journal vol.12 no.1 pp.331 -338, 1993

Functional role of proline and tryptophan residues highly conserved among G protein-coupled receptors studied by mutational analysis of the m3 muscarinic receptor Jurgen Wess1, Sujal Nanavati, Zvi Vogel and Roberto Maggio National Institute of Neurological Disorders and Stroke, Laboratory of Molecular Biology, Building 36, Room 3D-02, Bethesda, MD 20892, USA 1Corresponding author Communicated by R.Henderson

Most G protein-coupled receptors contain a series of highly conserved proline and tryptophan residues within their hydrophobic transmembrane domains (TMD I-VII). To study their potential role in ligand binding and receptor function, the rat m3 muscarinic acetylcholine receptor was used as a model system. A series of mutant receptors in which the conserved proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, was created and transiently expressed in COS-7 cells. [3H]Nmethylscopolamine ([3H]NMS) saturation binding studies showed that three of the seven mutant receptors studied (Pro242 - Ala, TMD V; Pro505 - Ala, TMD VI; Pro540 - Ala, TMD VII) were expressed at 35-100 times lower levels than the wild-type receptor while displaying 'm3-like' antagonist binding affinities. Pro201 - Ala (TMD IV) showed drastically reduced binding affinities (up to 450-fold) for both muscarinic agonists and antagonists. Whereas most mutant receptors retained strong functional activity, Pro540 - Ala (TMI VII) was found to be severely impaired in its ability to stimulate carbachol-induced phosphatidyl inositol hydrolysis (E. - 25% of wild type m3). Interestingly, this mutant receptor bound muscarinic agonists with 7- to 19-fold higher affimities than the wild type receptor. The Trp - Phe substitutions (Trpl92 - Phe, TMD IV; Trp5O3 - Phe, TMD VI; Trp530 - Phe, TMD Vi) resulted in less pronounced changes (compared with the Pro - Ala mutant receptors) in both ligand binding and receptor function. Our data indicate that the proline residues that are highly conserved across the entire superfamily of G proteincoupled receptors play key roles in receptor expression, ligand binding and receptor activation. Key words: G protein-coupled receptors/ligand binding site/muscarinic receptors/phosphatidyl inositol hydrolysis/ site-directed mutagenesis

Introduction A large number of cell surface receptors induce their intracellular responses via coupling to guanine nucleotidebinding regulatory proteins (G proteins). During the last decade, numerous members of this superfamily of G proteincoupled receptors have been cloned and sequenced (for recent reviews, see Strosberg, 1991; Dohlman et al., 1991; Oxford University Press

Probst et al., 1992). The ligands that can activate these receptors are remarkably diverse and include, for example, the classical biogenic amine neurotransmitters (e.g. acetylcholine, noradrenaline or dopamine), small peptides, glycoprotein hormones, odorants and light-activated retinal. Despite the structural diversity of the activating ligands, all G protein-coupled receptors share a common topographic motif consisting of seven hydrophobic transmembrane domains (TMD I -VII, predicted to form a-helices) that are joined by alternating intracellular and extracellular loops. The high degree of conservation of this structural arrangement throughout evolution suggests that it is particularly well suited for transmitting extracellular signals across the plasma membrane. A considerable number of studies with biogenic amine receptors (Hulme et al., 1990; Dohlman et al., 1991; Strosberg, 1991), the retinal pigment rhodopsin (Findlay and Pappin, 1986; Hargrave and McDowell, 1992) and the structurally related photoreceptor protein, bacteriorhodopsin (Findlay and Pappin, 1986; Henderson et al., 1990), suggest that ligand binding occurs within a cavity enclosed by the seven TM helices. The ensuing conformational changes within the helical bundle are then transmitted to the cytoplasmic face where the interaction with specific G proteins is thought to occur. Sequence alignments have shown that virtually all G protein-coupled receptors share, besides their common transmembrane disposition, 20 invariant amino acids, most of which are located within the seven TM helices (Findlay and Eliopoulos, 1990; Hibert et al., 1991; Probst et al., 1992). The high degree of conservation of these residues suggests that they may play key roles in protein folding and/or receptor function. Initial studies have focused on a series of conserved cysteine (Dixon et al., 1987; Fraser, 1989; O'Dowd et al., 1989; Dohlman et al., 1990; Kurtenbach et al., 1990) and aspartate residues (Fraser et al., 1988, 1989; Strader et al., 1988) and have delineated important functional roles for some of these amino acids. In contrast, little is known about the functional significance of a series of membrane-embedded tryptophan and proline residues that are particularly well conserved among all G protein-coupled receptors (Figure 1). Despite the lack of direct experimental evidence, several authors have suggested that the presence of the conserved proline residues, which are likely to introduce kinks into the TM helices (Findlay and Eliopoulos, 1990), may be essential for receptor function. It has been proposed for example that the presence of both 'straight' and 'angled' TM helices may be important to allow relative movements within the helical bundle necessary for receptor activation (Hulme et al., 1990). On the other hand, computer-assisted molecular modeling techniques predict that some of the conserved tryptophan residues may be directly involved in ligand binding (Hibert et al., 1991). To provide direct experimental evidence for the functional importance of the conserved proline and tryptophan residues, 331 -

J.Wess et al.

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Intracellular Fig. 1. Model of the seven transmembrane domains (TMD I-VIH) of the rat m3 muscarinic receptor. The numbers given below the individual amino acids refer to their position within the rat m3 receptor sequence. The proline and tryptophan residues highlighted in black are found in almost all G protein-coupled receptors. Trp530 (W530 in TMD VII; stippled circle) is highly conserved only among the biogenic amine and tachykinin receptors, but is usually absent in other G protein-coupled receptors. All highlighted proline and tryptophan residues were individually replaced with alanine and phenylalanine, respectively, as described in Materials and methods. The boxed residues have been shown to be critically involved in the binding of muscarinic ligands (Curtis et al., 1989; Fraser et al., 1989; Kurtenbach et al., 1990; Wess et al., 1991). With the exception of Aspl47 (TMD III) and Tyr533 (TMD VII), which are present in all biogenic amine receptors, these residues are conserved only within the muscarinic receptor family (ml -im5). The single letter amino acid code is used.

we have employed site-directed mutagenesis, using the rat m3 muscarinic acetylcholine receptor as a model system. The conserved proline residues were individually replaced with alanine (a good helix former that should lead to a 'straightening' of the TM helices), whereas the conserved tryptophan residues were converted into phenylalanine. The ligand binding and functional properties of the resultant mutant receptors were studied following their transient expression in COS-7 cells. Whereas less pronounced changes in receptor function resulted from the Trp - Phe substitutions, drastic changes in receptor density (Bmax), ligand binding affinities and functional activity were observed with the various Pro - Ala mutant m3 receptors. Our data suggest that the conserved proline residues may play key roles in the function of all G protein-coupled receptors.

Results To study the functional roles of a series of tryptophan and proline residues that are found in almost all G proteincoupled receptors (Figure 1), we have individually replaced these amino acids with phenylalanine and alanine, respectively, using the rat m3 muscarinic receptor as a model system. The agonist and antagonist binding properties of the resultant mutant receptors as well as their ability to stimulate phosphatidyl inositol (PI) hydrolysis were determined following their expression in COS-7 cells. Trp530 (located in TMD VII, Figure 1) which is only conserved among the biogenic amine and tachykinin receptors was also included in this study. Bmax levels and Northern analysis Transfected COS-7 cells were first examined for their ability to specifically bind the muscarinic antagonist, [3H]Nmethylscopolamine ([3H]NMS). Whereas no binding activity was found with non-transfected cells, specific [3H]NMS binding was observed after transfection with the wild-type and all mutant m3 receptor cDNAs. However, [3H]NMS saturation binding assays revealed dramatic

332

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Fig. 2. Northern blot analysis of total RNA isolated from COS-7 cells transfected with wild type and mutant m3 receptor cDNAs. RNA (- 10 /.g/lane) was electrophoresed, blotted and hybridized as described in Materials and methods. A 32P-labeled 0.7 kb m3 cDNA restriction fragment was used as a probe to detect m3 receptor-specific rnRNA levels. As an internal control, the same blot was reprobed with a 32P-labeled 3-actin cDNA probe. Trp5O3 - Phe and Trp 530 Phe were not included in this experiment, since both constructs yielded relatively high receptor levels (Bm.). Specific mRNA bands were quantified by computer-assisted densitometry of autoradiograms. Levels of receptor mRNAs were normalized to amounts of ,B-actin mRNA present in each lane. The normalized levels of mutant m3 receptor mRNAs were (wild type m3 = 100%): Trpl92 - Phe (30%), Pro201 -Ala (32%), Pro242 - Ala (15%), Pro5O5 -Ala (153%), and Pro540 - Ala (40%). The experiment was performed twice with similar results.

differences in maximum receptor numbers (Bmax) found with the different mutant receptor constructs (Table II). Receptor expression levels were found to be high for wildtype m3, Trp5O3 - Phe, Trp530 - Phe, and Pro201 Ala, intermediate for Trpl92 - Phe, and remarkably low (- 35- to 100-fold lower than for wild type m3) for Pro242 - Ala, Pro5O5 - Ala, and Pro540 - Ala. To test the possibility that the very low Bmax values seen with three of the four Pro - Ala mutant receptors resulted from decreased mRNA levels (e.g. due to differences in mRNA stability or rate of transcription), Northern analysis of total RNA isolated from COS-7 cells transfected with 20 tg of plasmid DNA was performed. Specific m3 receptor mRNA bands of the expected size (-4 kb) were detected

Role of conserved proline and tryptophan residues Table I. Ligand binding properties of mutant m3 muscarinic receptors expressed in COS-7 cells Receptor

m3 (wild type)

Trpl92- Phe Trp5O3- Phe Trp530 -Phe Pro201 - Ala Pro242-Ala Pro5O5 -Ala Pro540 - Ala

Antagonist binding [3H]NMS KD (pM) 36 74 177 40 6300 69 37 49

+ 5 ± 11 10 4 2 ± 770 4 ± 1 ± 5

4-DAMP 0.42 1.9 10 0.21 187 0.98 0.74 0.38

Ki

(nM)

± 0.03 0.3 1 ± 0.03 + 9 0.02 ± 0.04 ± 0.01

Agonist binding Acetylcholine Kapp (AM) 0.88 12 16 0.72 331 30 3.1 0.13

± 0.01 2 1 ± 0.07 ± 13 1 ± 0.4 ± 0.04

Carbachol app (JLM) 0.64 0.72 0.68 0.81 0.96 0.88 0.90 0.98

+ 0.04 +

± ± ± ±

0.08 0.03 0.01 0.05 0.04 0.01 0.04

5.1 35 41 2.8 395 80 13 0.27

± 0.4 6 10 ± 0.9 ± 50 3 + 5 4 0.10

0.67 0.67 0.69 0.81 0.80 0.61 0.80 0.82

+ 0.01

0.04 ± 0.07 ± 0.05 ± 0.11 0.01 ± 0.03 ± 0.05

Binding parameters were determined in direct ([3H]NMS) or in competition binding studies (all other ligands), as described in Materials and methods. Hill coefficients (nH) were not significantly different from unity in the antagonist binding studies, but were usually significantly smaller than 1 (P < 0.05) in the agonist binding assays. Ka values were calculated from IC50 values using the Cheng-Prusoff equation (Cheng and Prusoff, 1973) and can be considered approximations of values. Data are presented as means ± SEM of 2-4 independent experiments, each performed in duplicate.

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after transfection with the wild type and the various mutant receptor genes studied (Figure 2). With the exception of Pro5O5 - Ala, mRNA levels found with the mutant receptor constructs proved to be 2-7 times lower than the observed wild type m3 mRNA levels. The precise reason for this variation in mRNA levels remains unknown. One may speculate that slight differences in transformation efficiency and/or mRNA stability contribute to this phenomenon. Although transfection with Pro201 - Ala cDNA resulted in - 3-fold reduced mRNA levels (compared with wild type m3), the Bmax values found with this mutant receptor were similar to those observed with the wild type receptor (Table II). In addition, transfection with Pro5O5 - Ala cDNA yielded mRNA levels that were even higher (- 1.5-fold) than the corresponding wild type levels, whereas the number of detectable [3H]NMS binding sites was reduced by a factor of - 35 (compared with wild type m3). These observations suggest that the strong reductions in B,,.x values found with Pro242 - Ala, ProSO5 - Ala, and Pro540 - Ala cannot simply be explained based on differences in mRNA levels. Ligand binding studies The agonist and antagonist binding properties of the various mutant m3 receptors are summarized in Table I. Trp 192 Phe and Trp5O3 - Phe displayed clearly reduced binding affinities (up to 24-fold lower than the wild type receptor) for both muscarinic antagonists [[3H]NMS, 4-diphenylacetoxy- N-methylpiperidine methiodide (4-DAMP)) and agonists (acetylcholine and carbachol) (Table I, Figure 3A and B). In contrast, Trp530 - Phe bound all investigated muscarinic ligands with affinities that were similar to or even slightly higher than those found with the wild type receptor (Table I, Figure 3A and B). Most strikingly, Pro201 - Ala exhibited drastically reduced affinities ( - 80- to 450-fold) for both muscarinic agonists and antagonists (Table I, Figure 3C and D). In contrast, Pro242 - Ala, ProSO5 - Ala, and Pro540 - Ala displayed 'm3-like' antagonist binding affinities, but clearly differed in their agonist binding profiles. Pro242 - Ala bound the agonists acetylcholine and carbachol with 16- to 34-fold reduced affinities (compared with wild type m3), whereas only a slight decrease in agonist binding affinities (- 3-fold) was found with ProSO5 - Ala. Interestingly, Pro540 - Ala gained the ability to bind acetylcholine and

carbachol with exceptionally high affinities, which exceeded the corresponding wild type affinities by 7- and 19-fold, respectively (Table I, Figure 3D). To investigate whether Pro540 - Ala corresponds to a receptor in a permanent high affinity state or to a receptor permanently coupled to G proteins, carbachol binding studies were carried out in the absence and presence of the hydrolytically stable GTP analog 5'-guanylyl imidodiphosphate [Gpp(NH)p] (100 AM). High affinity agonist binding to Pro540 - Ala appeared to be independent of G protein coupling, since addition of Gpp(NH)p had no significant effect on carbachol binding to this receptor (without Gpp(NH)p: IC50 = 1.40 4 0.09 AM, nH = 0.87 i 0.05; with Gpp(NH)p: IC50 = 1.32 -fi 0.12 AtM, nH = 0.89 + 0.03; n = 2). However, as frequently found with PI-coupled muscarinic receptors (Shapiro et al., 1988; Wess et al., 1990, 1991), Gpp(NH)p treatment also did not significantly affect carbachol binding to the wild type m3 muscarinic receptor [without Gpp(NH)p: IC50 = 34 + 2 AM, nH = 0.69 + 0.04; with Gpp(NH)p: IC50 = 36 ± 4 AtM, nH = 0.72 + 0.03; n = 2], thus limiting the conclusions that can be drawn from these experiments. Stimulation of PI hydrolysis To determine whether the various mutant receptors, when expressed in COS-7 cells, remained functionally active, their ability to stimulate PI hydrolysis upon incubation with the agonist carbachol was determined. Since several of the mutant receptors were expressed only at very low levels, preliminary studies were carried out with the wild type m3 receptor to determine the effect of receptor density on receptor-mediated PI hydrolysis. As shown in Table II, an almost proportional reduction in Bma, values (as measured in [3H]NMS saturation binding studies) was observed after stepwise reduction of the amount of transfected wild type m3 expression plasmid (Rm3pcD). Interestingly, maximum PI hydrolysis (measured as accumulation of inositol monophosphate in the presence of 10 mM LiCl) proved to be unaffected by changes in m3 receptor numbers (Table II, Figure 4). However, the reduction in Bmax values resulted in a progressive rightward shift of the carbachol concentration-response curves (loss in carbachol potency)

(Table II, Figure 4). All three Trp - Phe mutant receptors studied retained 333

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Fig. 3. Displacement of specific [3H]NMS binding to Trp - Phe (A and B) and Pro- Ala (C and D) mutant m3 muscarinic receptors by the antagonist 4-DAMP (left) and the agonist acetylcholine (right). Competition binding studies were carried out with membrane homogenates prepared from transfected COS-7 cells as described in Materials and methods. The various symbols represent the following receptors: (A and B), wild type m3 (0), Trpl92- Phe (O), Trp5O3 - Phe (A) and Trp530 - Phe (A); (C and D), wild type m3 (0), Pro201 - Ala (V), Pro242 - Ala (V), ProSOS -Ala (*), and Pro540-Ala (o). The binding curves for the wild type m3 receptor are shown in bold. Actual binding parameters are given in Table I. Binding curves were generated by computer fit as described (Dorje et al., 1991). Each curve is representative of two to four independent experiments each performed in duplicate.

Table II. Bmax values and functional properties of mutant m3 muscarinic receptors expressed in COS-7 cells Receptor m3 (wild type) m3 (wild type) m3 (wild type) Trpl92 - Phe Trp5O3 -Phe Trp530 - Phe Pro201 - Ala Pro242- Ala Pro5O5 -Ala Pro540- Ala

DNA transfected

Bmax

Carbachol

(/g)

(fmol/mg)

EC50 (JLM)

Maximum increase in IP, levels above basal (%)

0.5 2 20 20 20 20 20 20 20 20

44 140 1630 220 930 1410 1420 16 41 47

± 0.1 ± 1.8 ± 0.06 ± 0.2 ± 2.0 ± 0.09 ± 16 ±1 ± 0.4 0.51 0.12

198 203 199 210 134 142 202 134 202 48

± ± ± ± ± ± ±

8 15 450 40 320 370 480

±4 ± 6 7

4.1 2.6 0.21 2.4 5.1 0.18 177 17 2.5

2 2 27 47 38 36 23 41 ± 22 3

± ± ± ± ± ± ±

Bmax values were determined in [3H]NMS saturation binding studies and indicate the maximum number of binding sites/mg of membrane protein. To obtain wild type Bmax levels that were as low as those found with some of the mutant receptors, the amount of transfected DNA (Rm3pcD) per 100 mm dish was reduced stepwise. In these experiments, vector DNA (without m3 cDNA insert) was added to the transfection mixtures to ensure that equal amounts of DNA (20 Ag) were present in each sample. EC50 values for carbachol-induced IP, accumulation were determined graphically from plots of log carbachol concentration versus percentage response. Data are presented as means ± SEM of 3-4 independent experiments, each performed in duplicate.

considerable functional activity (Table II, Figure 5A). Trpl92 Phe induced a maximum accumulation of inositol phosphates similar to the wild type receptor, whereas the maximum functional responses found with Trp5O3 Phe 70% and Trp530 - Phe were somewhat decreased (E. of that of wild type m3). Trpl92 - Phe and Trp5O3 - Phe -

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displayed clearly reduced carbachol potencies (Table II, Figure 5A), which paralleled the reductions in agonist affinities seen in the radioligand binding studies (Table I). Ala As shown in Figure 5B, three of the four Pro Ala, mutant receptors studied (Pro201 Ala, Pro242 and Pro5O5 Ala) retained strong functional activity. -

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Role of conserved proline and tryptophan residues

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Fig. 4. Effect of wild type m3 receptor number (Bmax) on carbacholinduced PI hydrolysis. COS-7 cells were transfected with 20 ug (*), 2 jig (0), or 0.5 Ag (U) of Rm3pcD plasmid DNA (Bonner et al., 1987), as described in Materials and methods. When