Escola Paulista de Medicina, UNIFESP, Sao Paulo, Brazil and 1 BIOcomputing, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany
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Abstract |
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Keywords: GPCR/G protein interaction/model
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Introduction |
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Several three-dimensional structures of G proteins have been determined (Noel et al., 1993; Lambright et al., 1996
; Sunahara et al., 1997
) and the receptor binding site of these proteins has been mapped using alanine scanning and functional assays (Hamm et al., 1988
; Onrust et al., 1997
; Kallal and Kurjan, 1997
; Grishina et al., 1998; Marsh et al., 1998
). For GPCRs, on the other hand, only low resolution structure data (Schertler and Hargrave, 1995
; Schertler et al., 1998) is available, but hundreds of mutation studies have been reported indicating that the cytosolic domains of G protein-coupled receptors (GPCRs) contain the sequence elements that are crucial for G protein coupling (for example, O'Dowd et al., 1988
; Franke et al., 1990
; Ohyama et al., 1992
; Namba et al., 1993
; Ernst et al., 1995
; Acharya et al., 1996, 1997
; Daaka et al., 1997
; Kostenis et al., 1997
; Lu et al., 1997
; Murthy et al., 1997; Olah et al., 1997; Sano et al., 1997
; Verrall et al., 1997
; Wess, 1997
; Wess et al., 1997
).
The early explanation for the GPCRG protein interactions was the so-called `one receptorone G protein' hypothesis that was proposed more than a decade ago when only rhodopsin and ß-adrenoceptors were well understood (Dohlman et al., 1987). In the last few years this hypothesis was silently extended to include that each G protein requires its own special set of cytosolic loops for proper binding (Dohlman et al., 1991
; Strader et al., 1994
).
A plethora of GPCRs but only a relatively small number of G proteins have been discovered (Horn et al., 1998), which required the introduction of the concept of promiscuity of G proteins. This promiscuity has been observed experimentally under physiological conditions as well as in heterologous expression systems (Ashkenazi et al., 1987
; Cotecchia et al., 1990
; Haga et al., 1990
; Chabre et al., 1992
; Eason et al., 1992
; Prather et al., 1994
; Kenakin, 1995
; Herrlich et al., 1996
; Laugwitz et al., 1996
; Clawges et al., 1997
) and the one-on-one hypothesis is no longer considered valid. Paradoxically, the idea that certain residues in the cytosolic loops are responsible for G protein specificity is still generally accepted. That this idea is wrong was demonstrated by sequence analyses which showed that significant correlations between sequence motifs in the cytosolic loops and a preference for certain G proteins can only be detected in a very few cases (Wenden,E.M., Oliveira,L., IJzerman,A. and Vriend,G., manuscript submitted). It is becoming increasingly clear that GPCRG protein coupling is regulated by cellular mechanisms (Neer, 1995
). The most likely cellular component to be involved is the cytoskeleton [for a review see Neubig, 1994] but the lipid composition (Harder et al., 1997) and proteins like those of the RGS family (regulators of G protein signalling; Sato et al., 1995; Druey et al., 1996; Koelle and Horvitz, 1996; Siderovski et al., 1996; Chen et al., 1997) etc., have all been invoked as factors which regulate G protein-coupling. Cytoskeletal proteins can regulate the efficacy and selectivity by keeping the components organized in compartments (Barroso et al., 1994; de Weerd et al., 1997; Cote et al., 1997
; Giesberts et al., 1997
; Hamilton et al., 1997; Hasegawa et al., 1997
; Saunders and Limbird, 1997
; Wozniak et al., 1997
; Oldenhof et al., 1998
; Saunders et al., 1998
).
The broad promiscuity of GPCRG protein coupling can only result from interactions between conserved parts of the receptor and conserved parts of the G protein that are complementary in their physicochemical characteristics. The alternative hypothesismany different contacts leading to the same final effectrequires too many fortuitous events to be realistic. We assume that the interactions between these conserved regions provide the major part of the binding energy. Other interactions, the local concentration of the partners in the coupling process, and cellular factors fine-tune this binding.
We propose a model for the class A GPCRG protein interaction and G protein activation that is based on three important facts:
These facts, combined with an extensive sequence analysis of both class A GPCRs (AGPCRs) and G proteins lead us to suggest a model in which the receptor triggers GDP release by disturbing the N- and C-terminal regions of the -subunit of the G protein. The model quite naturally explains the promiscuity of the AGPCRG protein interaction because interactions between the conserved parts of the receptor and conserved parts of the G protein take care of the binding.
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Material and methods |
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The average pairwise sequence identity in the AGPCR family is below 25% so normal multiple sequence alignment programs cannot be used to align this family. However, AGPCRs are highly conserved at a few positions (Figure 1). We therefore wrote a dedicated profile-based GPCR alignment program that aligns these highly conserved sequence motifs. The receptor sequences of the AGPCR family were aligned using profiles extracted from the GPCRDB (Horn et al., 1998
). In some cases these profiles were extended to align larger parts of the inter helical loops. This method has been described before in greater detail (Oliveiro et al., 1993).
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The AGPCR and G protein numbering systems used here have been described in Oliveira et al. (1993) and Lambright et al. (1996), respectively. All residues mentioned in this article are AGPCR residues, unless it is explicitly stated that they are part of the G protein.
Correlated mutation analysis
In the analysis of multiple aligned sequences one often comes across residue pairs or groups of residues that either remain conserved or mutate in tandem. Such pairs or groups of residues are said to show correlated mutational behaviour. The analysis of phylogenetic trees has been suggested as a tool to detect such groups, but correlated mutation analysis (CMA) is a more simple and more general tool for this purpose. The CMA techniques has been discussed extensively Kuipers et al. (1995). It has been shown that residues detected by the CMA method most often have a functional role, or are involved in intermolecular interactions Gobel et al. (1994).
Structure of AGPCRs
GPCRs form a large superfamily of receptors. They are presently divided into six main classes (Horn et al., 1998). These classes share no sequence similarity, and even within these classes the sequence similarities are often difficult to detect. Nevertheless, all GPCRs are expected to possess the same structural motif that is seen in the low resolution structures of rhodopsins (Schertler et al., 1993
, 1995): a transmembrane seven-helix bundle connected by three extracellular and three cytosolic loops, and N- and C-terminal domains located at the extracellular and cytosolic sides of the membrane, respectively. We concentrate on the AGPCR family because of the large number of sequences and the large amount of experimental data that is available for these sequences (Horn et al., 1998
).
Structure of G proteins
G proteins are heterotrimers consisting of -, ß- and
-chains. Many high resolution structures of G proteins or their components have been determined (Figure 2
). G
chains have the same fold as the small GTP-binding proteins (
ß-doubly wound fold), with an inserted helical domain (Noel et al., 1993
; Lambright et al., 1996
; Sunahara et al., 1997
). The Gß-chain forms a ß-propeller of seven WD-repeats. Gß is usually found in a complex with the
-chain (Lambright et al., 1996
). In the resting state the G protein trimer is in the GDP-bound form. Interaction with the receptor dissociates the G
chain from the ß
complex and leads to GDP release and GTP uptake (Sprang, 1997
).
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A model for GPCRG protein coupling |
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Here we mainly discuss the first four steps that all involve parts of the triggering mechanism. We don't intend to present any fine details about atomic interactions involved in this process, but rather present a mental model that helps one to think about the process.
The main consideration that went into the model was that the conserved cytosolic regions of AGPCRs interact with conserved parts of the G protein (which overlap with the experimentally determined receptor binding site on the G chain).
The conserved Arg340 residue of the DRY sequence motif is an important factor leading to GDP release. Arg340 interacts with a conserved negative residue in helix 5 of G
(Figure 3
). This negative residue, G
Asp337, is part of the arginine binding site. Disturbance of this site leads to release of the nucleotide.
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The following sections will discuss the computations and the experimental evidence found in the literature that lead us to this model. These topics are dealt with in the same order as presented here. A sequence analysis reveals the conserved residues in AGPCRs and G proteins. A CMA study hints at a binding pocket on the G chain. Experimental and theoretical studies confirm the important role of Arg340. Experimental evidence for the importance of the arginine binding pocket around Asp337 in the G protein is presented, and it shows that disruption of the structural integrity of this pocket leads to the release of GDP. Experimental evidence is presented for the importance of a helical part of the AGPCR C-terminal domain and for the N- and C-terminus of the G
chain. The interaction between these three structural elements is discussed. Experimental evidence for the importance of Tyr734 is presented and the role of this residue in stabilizing the active state of the receptor is discussed. Finally the role of the Gß
complex is discussed, and the model will be placed in a wider context.
Sequence analysis
GPCR sequence analysis.
Figure 1 summarizes the sequence analysis of AGPCRs. It can be seen that each helix contains at least one very conserved residue that can be used in the sequence alignment. This alignment is further aided by conservation of the loop length of the first two cytosolic loops.
Two features jump out at the cytosolic site: Arg340 and Tyr734. These residues are essentially 100% conserved and thus extremely likely to have an important functional role. Arg340 is the middle residue of the so-called DRY motif and Tyr734 is part of the NPXXY motif. Arg340 is the most conserved feature of the GPCRs. Only a few exceptions are known at this position (e.g. Cys340 in prostaglandin D2 receptors, Gln340 in one olfactory-like receptor subtype and Thr340 in the equine herpesvirus 2 receptor). The NPXXY motif is conserved in 94% of all AGPCRs. In a further 3% the motif is NPXXXY. In the remaining 3%, NPXXF is observed most often (e.g. the prostanoid receptors ipr and ep2). We have no idea about the structural or functional implications of these exceptions, but as there are so few, we will not elaborate on this topic.
Figure 1 shows a few more conserved features like a series of hydrophobic residues shortly after the DRY motif, a tyrosine near the cytosolic end of helix V and an FR motif about 15 residues after helix VII.
Multiple sequence alignments for AGPCRs as well as for GPCR families, sub-families, etc., can be found at http://www.gpcr.org/7tm/
G sequence analysis
A multiple sequence alignment of G chains reveals about a dozen conserved residues. Almost all these conserved residues play a role in GDP/GTP binding. A CMA analysis on the aligned G
chains reveals several groups of residues that during evolution remained conserved as a group, or mutated as a group. One group of positions displaying correlated behaviour (Table I
) involves mainly residues in the loop ß2-ß3 and in the helices
5 and
N.
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Highly conserved residues normally have a functional role in proteins, and the function that is common to all GPCRs is that the signal has to be transduced to the G protein. This led us to hypothesise in 1994 (Oliveira et al., 1994) that Arg340 sits in a polar pocket between the helices in the inactive form of the receptor. Upon activation the arginine moves out and sticks into the cytosol. Most of the reasoning in this 1994 article is still valid today, but more importantly, this arginine switch mechanism has now been validated experimentally (Acharya et al., 1996; Lu et al., 1997
) and theoretically (Scheer et al., 1996
). Scheer et al. (1996) made a molecular dynamics simulation of the
1-adrenoceptor wild type and mutants whereas Acharya and Karnik (1996) experimentally produced similar mutations in the D(E)RY motif of rhodopsin. They observed similar effects for mutations at position 339. Although these two groups draw quite different conclusions from their mutation studies, they agree on the fact that Arg340 is the residue that determines whether the receptor is in the R* or in the R state. Scheer et al. (1996) concluded that our earlier hypothesis that the Arg moves from a polar pocket between the helices to the cytosol upon activation was correct. Acharya and Karnik (1996) did not draw any structural conclusions, but stated that Arg340 is directly involved in the mechanism leading to GDP release (Acharya et al., 1996). Ernst et al. (1995) inverted the position of Glu339 and Arg340 in rhodopsin and observed impaired GDP release from transducin. The movement of the Arg340 side-chain has also been inferred from experiments performed on rhodopsin, because a light-mediated environmental change in the receptor allosteric site leads to an increase of the pKa of carboxylates, probably the Asp or Glu224 and Glu339 in the polar pocket between the helices (Arnis and Sakmar, 1993
; Arnis et al., 1994
). Cohen et al. (1993) and Shi et al. (1998) have observed in integral membrane preparations that the mutant Arg340Ala in rhodopsin partially abolished the transducin activating effect. Shi et al. (1998) concluded that Arg340 is only important for triggering full transducin activation in crude membrane preparations of bleached rhodopsin. These results seem to weaken the role of Arg340 in the signal transduction mechanism. However, similar experiments performed on detergent-extracted membrane proteins showed that those mutants are unable to activate transducin (Shi et al., 1998
). The combination of these experiments seems to reinforce another crucial aspect of our model, namely, the role attributed to external factors such as cytosolic proteins. It would be nice to know which are those cytosolic factors that compensate the absence of Arg340 for about 50% in the experiments of Shi et al. (1998).
The arginine binding site on the G protein
Figure 2 shows a ribbon representation of the G
ß
trimer. Gß and G
are coloured as a function of the secondary structure, but G
is coloured as a function of sequence conservation. In Figure 2
all residues in the G
catalytic domain are coloured red that are highly conserved while the corresponding residue in Ras P21 (De Vos et al., 1988
) is conserved less than 90%. If we assume that conserved residues have a function, then this colouring scheme indicates those residues that have a function in the G protein but not in Ras P21 (thus, the GDT/GTP binding residues are not highlighted). These residues are found prominently at the G
Gß interface, in the strand ß2, in the helical domain and in helix
5 (Figure 2
, top left). Of these regions with conserved residues only helix
5 is located in an area that has been determined experimentally to be involved in receptor binding. We therefore suggest that the direct partner for Arg340 in the receptor must be one of the two conserved aspartic acids residues in helix
5 (Asp333 or Asp337).
The N- and C-terminal regions of G chains form a compact domain in G
GDP complexes but become disordered upon activation (Mixon et al., 1995
). Deletion of the C-terminal 14 residues (337350 in the numbering system of reference 2; Figure 5
) in Go led to a loss of GDP affinity and to receptor independent G protein activation in the presence of GTP or GTP
S, but these effects were not seen after deleting the residues 341350. The explanation for these results was that the positions 338340 are occupied by conserved aliphatic residues that interact with hydrophobic residues of the N-terminal region of the G
chain (Denker et al., 1992
). This was in agreement with the old hypothesis that the process of G
activation involves dissociation of its termini thereby allowing the G
C-terminal domain to shift and to function as a lever that opens the GDP site (Denker et al., 1992
, 1995
).
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The steps describing the GPCRG protein coupling for the rhodopsintransducin system show that the only functions for the receptors are to bind the G protein trimer and to trigger a conformational change in the G chain that leads to GDP release. After that the GTP uptake triggers all the subsequent events including receptor dissociation (Acharya et al., 1997
). The simplest way for a receptor to perform this task would be to bind to a site on the G
chains (Figures 3 and 6
) such that the same effect is produced as by the deletion of 14 C-terminal residues as described above. We think that binding of the receptor's Arg340 to the G protein's arginine binding pocket leads to a destabilization of the helix
5 and the loop
5/ß6 in agreement with suggestions by others (Onrust et al., 1997
; Grishina et al., 1998; Marsh et al., 1998
; Tanaka et al., 1998
).
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Multi-helix interaction
The receptor side.
It has been shown experimentally that the C-terminus in the somatostatin 5, CCK and gonadotropin-releasing hormone receptors is involved in receptor internalization and G protein coupling (Go et al., 1998; Heding et al., 1998
; Hukovic et al., 1998
). In the angiotensin II receptors the C-terminus can be logically divided in two sections based on its functional properties (Thomas et al., 1995a
,b
):
The C-terminal residues that are involved in G protein binding are located just after the conserved FR motif at position 750751 (Sano et al., 1997). A peptide corresponding to this region in the angiotensin II receptor showed a high propensity to form
-helices in CD and NMR (Franzoni et al., 1997
; Yeagle et al., 1997
) experiments. Combined with the AGPCR sequence analysis which revealed insertions and deletions (which is indicative of a loop rather than of a regular secondary structure) between helix VII and the C-terminal domain, we modelled this receptor region as a short loop of five residues followed by an
-helix of about 15 residues that starts just before the conserved positions 750 and 751. In transducin the C-terminal end of this helix would correspond to a cysteine that is palmitoylated and inserted into the membrane (Khorana et al., 1992).
These assumptions disagree with the interpretation of results from recent EPR and kinetic studies on rhodopsin mutants containing Cys-substitutions in the C-terminal domain (Altenbach et al., 1999; Cai et al., 1999
). In these studies, the authors postulate a cytosolic extension of helix VII based on an
-helix-like periodicity of effects observed in an EPR study. However, these same authors showed in a previous publication (Yang et al., 1996
) that position 753 in rhodopsin is close enough to residue 140 in the cytosolic loop III to form a disulphide bridge when two cysteines are introduced at these positions. As stated by the authors (Altenbach et al., 1999
), this bridge cannot be formed if the rhodopsin C-terminus is an extension of helix VII, but can very well be formed if the protein chain would be bent by a loop after Tyr734.
The G protein side.
The only structural data for the C-terminal extreme end of the G chain was reported in the article by Noel et al. (1993). Unfortunately, helix
N was proteolytically deleted in this structure and the structure of the C-terminus was forced upon it by a large number of crystal contacts. Other G
structures show a well ordered helix
N but nothing beyond helix
5. By superposing both structures on the direct environment of helix
5, a `complete' hybrid G
structure can be obtained that indicates an antiparallel interaction between helix
N and the extreme C-terminal end.
Additional structural data for the extreme C-terminal end of the transducin G chain have been obtained from NMR studies (Dratz et al., 1993
; Kisselev et al., 1998
) but the conclusions of these studies were contradictory. This region has been claimed responsible for the GPCR specificity of G
chains (Conklin et al., 1993
; Acharya et al., 1997
; Kostenis et al., 1997
) and has been shown disordered when free, but forming a ß-turn in the C-terminal five residues when complexed with light-activated rhodopsin (Dratz et al., 1993
). Another study contradicts these results showing that the G
C-terminal peptide assumes a helical conformation in the presence of light-activated rhodopsin (Kisselev et al., 1998
). These authors model this helix as a continuation of helix
5 (Kisselev et al., 1998
). No matter which result is correct the structuring effect of activated rhodopsin on the G
chain is in all instances due to interactions with the receptor cytosolic loops (Kostenis et al., 1997
; Kisselev et al., 1998
) or with the DRY motif (Acharya et al., 1997
).
Multi helix binding.
We assume that the receptor C-terminal helix interacts with helix N in the G protein and perhaps also with the C-terminal 10 residues of the G protein. The structure of those 10 residues is unknown, and, as discussed above, many proposals have been made (Dratz et al., 1993
; Kisselev et al., 1998
). Our model could accommodate each of these proposals. However, we consider the proposal obtained from X-ray data the most logical one, despite the crystal packing artefacts. We thus suggest that the C-terminus of the G
chain becomes ordered upon receptorG protein coupling because of interactions with the N-terminus of the G
chain and the C-terminus of the receptor (Figure 6
).
This type of interaction would fit our model very well, because the helixhelix interactions can be achieved by many types of hydrophobic residues, without the need for specific residueresidue interactions. This arrangement forces the part of the receptor C-terminal domain that is beyond the helix far away from the Greceptormembrane complex which makes this part more accessible to kinases and other cytosolic regulatory proteins (Palczewski et al., 1997). This agrees well with the observation that this part of the sequence contains many phosphorylation sites.
The role of Tyr734
We modelled the active form of the receptor. In this model the Tyr734 side chain is placed at hydrogen bonding distance to the Arg340 guanidinium group (Figure 6), but we could just as well have modelled a hydrogen bond to Asp339. Some doubt exists about the role of Tyr734 in G protein signalling and in other processes such as desensitization and internalization, because much contradicting literature exists about the role of this residue. Tyr734 has been shown crucial for receptorG protein coupling in rhodopsins (Cai et al., 1999
),
1B-adrenoreceptors (Wang et al., 1997
) and angiotensin II receptors (Laporte et al., 1996
), and for receptor internalization in ß2-adrenoceptors (Barak et al., 1994
) and substance P receptor (Bohm et al., 1997
). On the other hand, Tyr734 has been shown to neither be important for internalization of angiotensin II receptors (Thomas et al., 1995a
,b
) nor for G protein coupling to ß2-adrenoceptors (Barak et al., 1994
).
Mutagenesis studies on 1B-adrenoreceptors showed that when Tyr734 is mutated, an increase in the ligand-receptor affinity occurs even when G protein coupling is abolished (Wang et al., 1997
). EPR studies revealed a change in the polarity of position 734 upon light-mediated receptor activation when this residue was mutated to cysteine (Altenbach et al., 1999
). These results point at the intriguing possibility that Tyr734 could be negatively regulating the agonist-receptor binding. If this is true, G protein coupling would favour agonist binding by moving Tyr734 away from the DRY region. There is not yet enough data available to speculate about a connection between the multi-helix interaction, Tyr734 and the stabilization of the MII state of rhodopsin.
It is tempting to speculate that the Tyr734 side-chain interacts with a residue in the DRY motif. The low resolution rhodopsin structure indicates that such an interaction is very well possible. When the experimental data listed above is combined with this assumption (that there is indeed an interaction between Tyr734 and the DRY motif) then there are two possible scenarios. One possibility is that the tyrosine interacts with the D or Y of the DRY in the inactive receptor (R state) and that the G protein breaks this interaction, thereby making it possible for Arg340 to move outwards and bring the receptor in the active (R*) state. The other possibility is that the G proteins binds to the receptor when Arg340 is pointing towards the cytosol and that this binding brings Tyr734 in a position where it interacts with Arg340, thereby stabilizing the R* state. This second hypothesis is more appealing because of its simplicity, but there exists more experimental data supporting the first hypothesis.
The role of Gß
Several mutation experiments have been performed on Gß (Ford et al., 1998
). Alanine scanning has been performed on those areas of the Gß
chains which were known from a study of the crystal structures to interact with the G
switch regions or with the N-terminal helix of G
(Lambright et al., 1996
). This study showed that the mutations Ile81Ala and Lys89Ala, located in the WD repeats 1 and 2 in Gß respectively, significantly reduce receptor coupling from which it has been concluded that the receptor must therefore make direct interactions with the Gß
subunits. As the residues at these positions are also important to hold the N-terminal helix of G
in place (Lambright et al., 1996
), we conclude that the main role of Gß
in receptor coupling is not to provide a binding surface for the receptor, but much more to help keep G
in the optimal conformation for receptor binding.
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Discussion |
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In the AGPCR model proposed by Baldwin et al. (1997) helix III in the receptor continues for two more turns after the DRY motif. As the first six residues after the DRY motif are highly conserved, it seems likely that they have a functional role (Farahbakhsh et al., 1995). NMR studies on isolated loops, however, suggest these residues to be in an extended conformation (Yeagle et al., 1997
). We cannot discriminate between these two alternatives, and within the low level of detail of our model, both alternatives would fit reasonably well, and all other contacts mentioned in this article agree with both alternative solutions for modelling the cytosolic end of helix III.
In our model the cytosolic loops don't have any detail. The loop structures determined by Yeagle et al. (1997) were determined in isolation and are unlikely to be very similar to the loops in the receptorG protein complex. Other structural information is unfortunately not yet available. We therefore modelled the loops as large half circles merely to show that there is ample space for these cytosolic loops to protrude out into the cytosol.
We have presented a very low resolution model for the GPCRG protein complex. In contrast to many models that are constructed using homology to a known three-dimensional structure, large parts of our model are inferred from non-structural experimental data. The model is far from complete. This model is in agreement with most of the available experimental data, but for several observations the model still suggests a very large number of solutions. A major problem in this respect is that the outcome of seemingly similar experiments is often contradictory, and authors often draw orthogonal conclusions from the same data (see Acharya and Karnik, 1996; Scheer et al., 1996 and Shi et al., 1998 for examples of beautiful, but nevertheless contradicting experiments).
We postulate that the GPCRG protein coupling is governed by interactions between conserved sequence elements. Although specificity is for a large part determined by other factors than GPCR sequence motifs, the model also explains the G protein specificity in the few classes where this can be attributed to sequence motifs (Wenden, E.M., Oliveira,L., IJzerman,A. and Vriend,G.). Our model also suggests a pathway for GPCRG protein signalling, and this pathway will be helpful in better understanding future experiments.
Our model is different from some other models in that we do not even try to arrive at `correct' three-dimensional coordinates for the complex. We merely try to indicate which residues could potentially interact. Our model is in many three-dimensional aspects similar to the recent model by Bourne et al., 1997; Onrust et al., 1997; Lichtarge et al., 1996; Iiri et al., 1998), with the difference that we try to show specific interactions at the receptor and G protein structures and relate them to specific steps in the coupling mechanisms of these proteins.
A different model for GPCRG protein coupling has recently been reported by Kisselev et al. (1999). Based on older observations that the C-termini of the G- (Dratz et al., 1993
) and G
-chain (Kisselev et al., 1994
) (the latter one farnesylated) can stabilize activated MII forms of rhodopsin, these authors proposed (without taking any other experimental evidence into account) a two-site sequential model for signal transduction (Kisselev et al., 1999
). Although interesting, this mechanism should be analysed with caution because it seems that the phosducin-mediated binding of the farnesylated C-terminus of the G
-chain to its natural site on the Gß-chain (Loew et al., 1998
) was not considered.
To the best of our knowledge, only one other model is available in the literature in which ideas are mapped on low resolution 3D coordinates. This model (Nederkoorn et al., 1998) focuses on the principle that GPCR acts as a proton pump and thus G protein coupling is a way to allow proton arrival to a certain region of the Gß chain followed by GTP regeneration from GDP analogous to the mechanism of synthases.
We believe in Occam's razor, and our model has been constructed keeping that idea (when two possibilities exist, the simplest should be chosen) in mind. The model cannot be free from exceptions because receptors and G proteins exist that lack some of the residues that are crucial in our model and the model does not provide three-dimensional coordinates, but it does provide a mental framework that can be used to think coherently about the large volume of presently available experimental data.
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Acknowledgments |
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Notes |
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References |
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Received March 3, 1999; revised August 27, 1999; accepted September 1, 1999.