Lipid-facing correlated mutations and dimerization in G-protein coupled receptors

Paul R. Gouldson1, Mark K. Dean1, Christopher R. Snell2, Robert P. Bywater3, Georgios Gkoutos1 and Christopher A. Reynolds1,4

1 Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, 2 Novartis Institute for Medical Sciences, 5 Gower Place, London WC1E 6BN, UK and 3 Biostructure Department, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
A correlated mutation analysis has been performed on the aligned protein sequences of a number of class A G-protein coupled receptor families, including the chemokine, neurokinin, opioid, somatostatin, thyrotrophin and the whole biogenic amine family. Many of the correlated mutations are observed flanking or neighbouring conserved residues. The correlated residues have been plotted onto the transmembrane portion of the rhodopsin crystal structure. The structure shows that a significant proportion of the correlated mutations are located on the external (lipid-facing) region of the helices. The occurrence of these highly correlated patterns of change amongst the external residues suggest that they are sites for protein–protein interactions. In particular, it is suggested that the correlated residues may be involved in either large conformational changes, the formation of heterodimers or homodimers (which may be domain swapped) or oligomers required for activation or internalization. The results are discussed in the light of the subtype-specific heterodimerization observed for the chemokine, opioid and somatostatin receptors.

Keywords: dimerization/G-protein/lipid-facing/mutations/rhodopsin crystal structure


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
G-protein coupled receptors (GPCRs) constitute a large family of homologous transmembrane proteins which are activated by a variety of different ligands such as hormones, neurotransmitters and calcium ions (Strader et al., 1994Go; Watson and Arkinstall, 1994Go; Wess, 1998Go). The possibility that GPCRs may dimerize, and that the dimer may be the functionally active form of the receptor has received scant attention until this was demonstrated in a series of experimental studies (Maggio et al., 1993Go; Hebert et al., 1996Go; White et al., 1998Go). This, together with the publication of the first X-ray crystal structure for a GPCR—rhodopsin (Palczewski et al., 2000Go) has prompted this sequence-based study of the external residues of various GPCR families.

The essential structural features of GPCRs are an extracellular N-terminus, a serpentine portion containing seven transmembrane (7TM) helices connected by intracellular and extracellular loops and an intracellular C-terminus. The transmembrane portion is believed to pack in a similar manner to that of rhodopsin (Baldwin et al., 1997Go) such that the hydrophobic faces of the helices point out towards the lipid while the interior is generally more hydrophilic. In a number of GPCR families (e.g. the biogenic amine receptors), the internal transmembrane residues are involved in ligand binding (Baldwin, 1994Go; Herzyk and Hubbard, 1995Go); in other families (e.g. neurokinin, glucagon) the N-terminus and the extracellular loops are also involved in ligand binding (Fong et al., 1993Go; Buggy et al., 1995Go) while in others (e.g. metabotropic glutamate) the ligand binds to the N-terminus (Kunishima et al., 2000Go). The nature of the external lipid-facing residues is generally considered unimportant as long as they are hydrophobic and this assumption often provides a very useful working hypothesis (Baldwin et al., 1997Go). However, the evidence presented from this correlated mutation analysis (CMA) of the aligned sequences of a number of class A receptors, namely the neurokinin, opiate, somatostatin, chemokine, thyrotrophin (TSH) and the whole amine family suggests that this view may mask some important features of GPCR structure and function.

CMA is a technique for the identification of highly correlated patterns of change amongst the aligned sequences of a set of proteins (Gobel et al., 1994Go; Singer et al., 1995Go; Pazos et al., 1997Go). The technique was greeted with much enthusiasm as a way of predicting residue–residue contacts within a protein core and therefore for predicting three-dimensional structure from sequence (Gobel et al., 1994Go; Benner, 1995Go; Ortiz et al., 1998Go). In many protein families this potential has not been fully realized (Taylor and Hatrick, 1994Go) but in the GPCRs, the technique has yielded interesting observations (Kuipers et al., 1997Go; Horn et al., 1998aGo), partly because the sequences are highly homologous, particularly in the transmembrane domains (typical values range from to 35% to over 95% similarity). In addition, there are a large number of available sequences (currently many hundreds: Horn et al., 1998bGo; Vriend, 2000Go), the receptors have appropriately developed dendritic trees and model structures can be generated by homology (Higgs and Reynolds, 2000Go) to the rhodopsin crystal structure (Palczewski et al., 2000Go; Teller et al., 2001Go) to assist in the interpretation of the results. Moreover, recent research (Pazos et al., 1997Go; Nilsson et al., 1999Go) has supported earlier findings (Oliveira et al., 1993Go; Horn et al., 1998aGo) that correlated residues tend to accumulate at protein interfaces.

Our initial CMA studies on the adrenergic, dopaminergic and muscarinic receptors revealed a number of correlated mutations amongst the external transmembrane residues (Gouldson et al., 1997aGo,1998Go), as summarized in Figure 1Go, and so here we have undertaken a more extensive analysis. The analysis has identified distinct correlations amongst the external residues, and one explanation is that these correlations may provide the molecular basis for the formation of protein–protein interactions. Consequently, some of the evidence for dimerization in receptor activation is presented. Some of the observed correlated mutations cannot be explained simply in terms of receptor dimerization and it is tentatively proposed that these residues may be involved in large conformational changes, higher order oligomer formation or in interactions with other as yet unidentified proteins.



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Fig. 1. External correlated mutations amongst (a) the dopamine and muscarinic receptors. The figure also shows the external correlated mutations amongst the (b) the adrenergic receptors and (c) neurokinin and (d) opiate receptors.

 

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The basic principle underlying CMA is illustrated in Figure 2Go which shows the residues at positions N1–N5, which are not necessarily sequential, for seven different hypothetical {alpha} and ß receptors (which may be further divided into subtypes 1 and 2). The residue at position N1 (W) is conserved and any mutations at this point are likely to result in a loss of function. However, at position N2 it is likely that any residue may be present as long as it is hydrophobic. At position N3 the residue is conserved amongst the {alpha} receptors and mutation at this point may result in loss of function. However, since mutation of this Phe to an Asn is correlated with a corresponding mutation from Leu to Phe at position N4, the residues at positions N3 and N4 are said to be correlated and the second mutation may be responsible for the regain of function lost by the first mutation. The residues at position N5 are correlated with the subtype, thus a Ser is observed in ß1 receptors while an Asn is observed in ß2 receptors. In Figure 2Go, perfect correlations are observed but in reality this is not always the case since deviations from ideality may arise for a number of reasons, including sequencing errors and species divergence. Thus, in this article we have reported correlations above the 0.9 threshold, as defined by the CMA correlation coefficient (Singer et al., 1995Go). The CMA was performed using the WHATIF molecular graphics software (Vriend, 1990Go). An alternative implementation (Taylor and Hatrick, 1994Go) builds in amino acid properties such as volume or hydrophobicity, which may be related to the underlying physical properties. However, this alternative approach has not been as successful and this may be because compensatory mutations do not necessarily occur in the protein core as alternative mechanisms for alleviating strain are possible (see for example, Lesk and Chothia, 1980Go; van Gunsteren and Mark, 1992Go; Hubbard et al., 1994Go; Horn et al., 1998aGo). The accession names for the neurokinin, opioid, somatostatin, chemokine and TSH receptor sequences used are given in Table IGo. Throughout this work, the order of the sequences corresponds to the order given in Table IGo. The results were plotted onto the rhodopsin crystal structure, PDB code 1F88 (Palczewski et al., 2000Go; Teller et al., 2001Go) using the sequence homology.



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Fig. 2. Hypothetical pattern of resides found at positions N1 to N5 for seven hypothetical {alpha} and ß receptors; the pattern of residues is used to illustrate the principle behind CMA.

 

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Table I. Accession names for chemokine, neurokinin, opioid, somatostatin and TSH sequences used in the CMA
 

    Results
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 Methods
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 Discussion
 Conclusions
 References
 
A total of 160, 107, 108 and 190 correlated mutations were identified for the chemokine, neurokinin, opiate and somatostatin receptors, respectively, of which 37, 35, 46 and 50 may be assigned as external and to lie within the transmembrane domain, as determined using the crystal structure. The sequence numbers are reported for the NK-1, µ-opioid, somatostatin S1, chemokine CCR2 and ß2-adrenergic receptor subtypes, but the bovine rhodopsin and universal numbering schemes are also used (Oliveira et al., 1993Go). For the latter, the most conserved residue of each helix is assigned the number 30 (a conversion table is available at the GPCRDB website: Vriend, 2000Go). The positions of these external correlated mutations are shown in Figure 3Go. The CMA identified several networks of correlated mutations for each receptor, largely corresponding to the different receptor subtypes. Thus, one network identified residues that correlate with the NK-1, NK-2 and NK-3 subtypes, another network identified residues which correlate with the NK-1 subtype but were conserved (or random) across the other subtypes. Thus to design a drug specific to the NK-1 receptor (rather than the NK-2 or NK-3 receptors), it may be useful to consider both sets of results. External residues which correlate with the NK-2 and NK-3 subtypes were also identified. Similar networks were found for the chemokine, opioid and somatostatin receptors and selected results given in Tables II–VGoGoGoGo.



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Fig. 3. Positions of the external correlated mutations in selected GPCRs: (A) chemokine, (B) neurokinin, (C) opioid, (D) somatostatin and (E) TSH. The correlated residues are shown in dark grey, the helices are numbered in (A) and are shown in alternating light shades of grey.

 

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Table II. The progression from conserved residues to correlated residues on descending the dendritic tree from the ß2-adrenergic receptors to the amine receptorsa

 

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Table III. Candidate residues for determining the subtype specific heterodimerization observed in the somatostatin receptora
 

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Table IV. Candidate residues for determining the subtype specific heterodimerization observed in the opioid receptora
 

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Table V. Candidate residues for determining the subtype specific heterodimerization observed in the chemokine receptora
 
The TSH receptor does not have subtypes, and only six sequences were analysed (from six species). Nevertheless, 41 correlated mutations were observed and 20 of these can be assigned to external residues. These are given in Figure 3EGo. The correlated residues for the whole family of biogenic amine receptors (which includes the adrenergic, dopaminergic, histamine, serotonin and muscarinic receptors) are given in Figure 4Go. The residues conserved in the biogenic amine family are also shown in this Figure and it can be seen that most of the conserved residues are either flanking or in very close proximity to the correlated residues. Table IIGo shows how the (external) conserved residues in the ß-adrenergic receptors become correlated residues as one proceeds back up the dendritic tree towards the biogenic amine family.



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Fig. 4. A snake diagram presentation of the CMA results for the biogenic amine receptors. This involved 130 aligned receptor sequences taken from the adrenergic (43 sequences), dopamine (16 sequences), muscarinic (19 sequences), histamine (9 sequences) and the serotonin (43 sequences) receptors. The correlated residues are shown in black circles; the conserved residues are shown in white circles. The orientation of the helices is shown in the helical-wheel-like diagram (lower left).

 

    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
Helix orientation: the assignment of external residues

The CMA has identified a large number of correlated mutations amongst the various receptor families, of which about a third have been assigned as external. This assignment to external residues may be viewed as tentative since high resolution crystal structures for the active forms of the individual receptors are not yet available and it is known that helices 3, 4, 5, 6 and possibly 7 undergo conformational change on activation (Zhang and Weinstein, 1993Go; Luo et al., 1994Go; Farahbakhsh et al., 1995Go; Gether et al., 1995Go,1997aGo, bGo; Altenbach et al., 1996Go; Javitch et al., 1997Go). However, it is clear that most of the correlated residues identified as external must remain so. Analysis of Figure 1Go shows that no rotation of helices 1, 2, 4 or 6 could bury all of the adrenergic, dopamine and muscarinic correlated mutations; a similar situation applies to the neurokinin, opioid, somatostatin and TSH results. Thus, while the lipid accessibility of some of the residues on helices 5 and 6 may change during activation, the changes are not sufficient to bury all of the external correlated mutations. As discussed elsewhere, there is the potential for marked changes on activation in the intracellular half of helix 7 (Fu et al., 1996Go; Gouldson et al., 1997bGo; Konvicka et al., 1998Go; Higgs and Reynolds, 2000Go), but as few correlated mutations were identified in helix 7, and even fewer below the key NPXXY motif, this does not affect our results.

In addition to the site-directed mutagenesis information which gives a strong indication of helix orientation, much useful data can also be obtained from studies on engineered zinc binding sites (helices 2, 3, 5 and 6), the substituted cysteine accessibility method (SCAM) (helices 3, 4, 5 and 7), site-directed spin labelling studies (helices 3, 4, 5 and 6) and FT-IR studies (helices 3 and 5) (Gouldson,P., Kidley,N.J., Bywater,R.P., Diaz,C., Reynolds,C.A. and Shire,D., 2001, submitted). The data on zinc binding to specifically engineered binding sites is probably the most pertinent to this study as it has been generated using the neurokinin-1 (Elling et al., 1995Go; Elling and Schwartz 1996Go) and the {kappa}-opioid (Thirstrup et al., 1996Go) receptors. The data provides distance constraints between helices 2 and 3 and helices 3 and 5 in the neurokinin NK-1 receptors and between helices 5 and 6 in the neurokinin NK-1 and {kappa}-opioid receptor. The crystal structure is consistent with this data, confirming that the orientation of these helices is probably similar in rhodopsin, the neurokin receptors and the opioid receptors—at least in the inactive form of the receptor (when zinc binds it usually functions as an antagonist and so prevents the receptor attaining the active conformation, but see Elling et al., 1999Go). It seems reasonable therefore to use the rhodopsin structure throughout this study. The key question is therefore whether the occurrence of external correlated residues has any functional significance.

The link between correlated mutations, conserved residues and function

A simple view of amino acid function would suggest that only the conserved amino acids are important for the basic function of the protein. However, many of the amino acids that are correlated within a family become conserved in a sub-family. For example, residue 312 in the adrenergic receptors may be Phe or Asn but is a conserved Asn in the ß-adrenergic receptors. Here, this correlated residue has a clear function through its role in the {alpha}–ß switch (Suryanarayana et al., 1991Go; Suryanarayana and Kobilka, 1993Go; Gouldson et al., 1997cGo). Table IIGo shows the progression from conserved to correlated on going back up the dendritic tree from the ß2-adrenergic, through adrenergic and catechol (adrenergic and dopamine) through to the biogenic amine receptors. Indeed, following this dendritic tree back down a different branch from the correlated residues will identify further external conserved residues. However, the patterns observed will vary from receptor to receptor. For example, positions of the correlated residues in the ß-adrenergic receptor are occupied by conserved residues in the muscarinic receptor. For many other correlated residues, the function is not yet as clear as it is for Asn312. For example, there are many other residues, such as Val81, Val117 and Ile278 in ß2-AR which correlate with the {alpha}–ß switch in the adrenergic receptors and yet they are removed from both the ligand binding site and regions of the receptor which may contact the G-protein. In the neurokinin receptors too, there are many internal residues which correlate with the NK-1/NK-2/NK-3 switch, e.g. Met291, which when mutated in a subtype specific manner change the binding properties of the endogenous ligands (substance P, neurokinin A and neurokinin B) towards those of the alternative subtype even though modelling studies shows they are positioned well away from the binding sites (Gouldson, 1998Go). They may modulate the agonist-induced conformational change through effects on helix–helix packing or they may exist as vestiges of an earlier function. Elsewhere we observed that while the agonist tends to bind to the conserved residues, such as the serines on helix 5, the antagonist tends to bind to the correlated residues such as Asn3122-AR numbering) (Gouldson et al., 1997bGo,cGo). Thus, while the conserved residues are involved in the basic function of the receptor, it is possible that the correlated residues are involved in the finer details of specificity (as knowingly or unknowingly used by the medicinal chemist in the design of specific antagonists).

The association of the correlated residues with the conserved residues is most readily seen in the CMA of the whole biogenic amine family of receptors, shown in Figure 4Go. Not only does this Figure show the correlated residues (internal and external) but it also shows that the conserved residues are often either adjacent in the sequence to a correlated residue or that they are directly above or below a correlated residue in the structure. This association between correlated residues and conserved residues is also shown in Table IIGo, which shows that a large proportion of the conserved residues in the ß2-adrenergic receptor becomes correlated on going back up the dendritic tree through the adrenergic receptors (where 50% remain conserved and 43% become correlated), the catechol receptors (where 40% remain conserved and 19% become correlated) to the biogenic amine receptors (where 20% remain conserved and 5% become correlated). The tendency of correlated and conserved residues to cluster together in space is similar to the clustering observed for evolutionary trace residues (Lichtarge et al., 1996Go), which in some respects are similar to the correlated residues (Gkoutos et al., 1999Go).

In the TSH receptors, the vast majority (21/24) of the correlated residues in the transmembrane domain are external. The explanation may be that variation is most readily tolerated amongst the external residues. An alternative explanation is that the external residues are important and hence the variation is correlated rather than random. For this receptor, it is difficult to distinguish between the two cases but we note that there are TSH external correlated residue positions on helices 5 and 6 for which mutation in other receptors shows an effect on function. The positions are 511 (Ji et al., 1994Go; Puffenberger et al., 1994Go), 515 (Kosugi and Mori, 1996Go), 519 (Hwa et al., 1995Go), 526 (Ji et al., 1995Go) and 616 (Tomic et al., 1993Go; Hebert et al., 1996Go); further details are given elsewhere (Dean,M.K., Higgs,C., Smith,R.E., Bywater,R.P., Snell,C.R., Scott,P.D., Upton,G.J.C., Howe,T.J. and Reynolds,C.A., 2000, J. Med. Chem., submitted).

Thus, there are cases where the correlated residues have a clear function but also many cases where the function of the correlated residues is presently unknown. Since site-directed mutagenesis is the primary method for determining residue function, the correlated residues ought to be considered as prime candidates for mutagenesis.

Possible roles for external correlated residues in dimerization

The function, if any, of the correlated residues on the external face of the helices is currently unknown. If the receptor only made non-specific interactions with the lipid we would not necessarily expect to observe external correlated mutations. Since it is generally assumed that the external faces are not involved in ligand binding, the most likely explanation is that they are involved in helix–helix interactions, either during conformational changes within the monomer or during oligomer formation.

Recent work (Oliveira et al., 1993Go; Pazos et al., 1997Go; Horn et al., 1998aGo) has shown that there is a tendency for correlated mutations to occur at domain and protein interfaces. Pazos in particular, in a study of 21 proteins, demonstrated that CMA can be used to identify correctly docked protein structures from many incorrectly docked structures. In our study of the major histocompatibility complex class II receptor, for which there is again a crystal structure, we found a distinct tendency for correlated residues to lie at domain interfaces (Nilsson et al., 1999Go). Therefore, the evidence is growing that correlated residues may indicate a functionally important protein interface.

Since there is currently much evidence in the literature regarding the possibility of dimer formation in the activation of GPCRs, the external correlated residues may be involved in the dimer interface. A full account of the evidence for GPCR dimers is presented elsewhere (Gouldson et al., 1998Go,2000Go); here we simply list some of the main pieces of evidence, particularly those that shed some light on the possible dimer interface. Thus, the results of Hebert et al. on the ability of a peptide derived from helix 6 to inhibit both dimerization and activation (Hebert et al., 1996Go) are particularly convincing since this provides evidence that helix 6 may be involved in the formation of the dimer interface. A similar, though preliminary study, by Ng et al. also suggests that helix 7 may play a key role (Ng et al., 1996Go). Immunological studies (Ciruela et al., 1995Go) suggest that intracellular loop 3 is less exposed in the dimer, so we are able to imply that helices 5 and 6 play a role in dimerization.

The chimeric receptor studies (Maggio et al., 1993Go,1996Go) provide some of the most compelling evidence for dimerization. Maggio constructed chimeric receptors where the A domain (including helices 1–5) was taken from the ß2-adrenergic receptor and the B domain (including helices 6 and 7) was taken from the muscarinic M3 receptor. These receptors and the alternative muscarinic–adrenergic chimeras were unable to bind ligand or to stimulate phosphatidylinositol hydrolysis. However, binding and activity were restored when the two chimeras were co-expressed. We propose that domain swapping, as shown in Figure 5Go, provides the most likely explanation of this functional rescue. Moreover, if this mechanism can take place in chimeric receptors, it can probably also take place in wild-type receptors, since the dynamic nature of GPCR domains is well known (Gudermann et al., 1997Go), though domain swapping may require some additional degree of unfolding (Schulz et al., 2000Go;Rousseau et al., 2001Go). Similar evidence for domain swapping can be derived from the studies of Monnot et al. on the co-expression (and hence functional rescue) of receptors each containing a separate fatal mutation (Monnot et al., 1996Go)—except here we conclude that domain swapping resulted in the formation of a 4,5-domain swapped dimer (see Gouldson et al., 1998Go) leading to the restoration of binding, as an intact 7TM bundle was regenerated. We conclude that activity was not restored because the functionally important extensions to helices 5 and 6 and the intracellular loops were in the wrong relative orientation.



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Fig. 5. The proposed domain swapping rearrangement. In (a), helices derived from monomer 1 are shown as black circles; helices derived from monomer 2 are shown as grey circles. In (b) the domain swapping is illustrated using Maggio's chimeric adrenergic (grey)–muscarinic (black) receptors. (These are symmetric chimeric heterodimers.) (c) Contact dimers.

 
The mechanism shown in Figure 5a and bGo results in transient interactions between helices 1 and 7 and implicates helices 5 and 6 in the dimer interface. The studies of Monnot et al. would also suggest a functional role for helix 4 but elsewhere we argue that the 5,6-domain swapped or contact dimer, as shown in Figure 5a and bGo is the major physiological dimer (Gouldson et al., 1997bGo). Therefore, this dimerization mechanism provides a possible explanation for both external conserved and external correlated residues. Some researchers may question whether hydrophobic residues alone are able to provide a highly specific dimer interface. In a study of helix–helix interactions in globular proteins, Chothia observed that half of these interactions were mediated by hydrophobic interactions alone (Chothia et al., 1981Go). More significantly, in the glycophorin A dimer, which involves two transmembrane helices, the L75IXXGVXXGVXXT87 motif (Lemmon et al., 1994Go) is responsible for dimerization. This motif is very sensitive to minor changes, such as mutation of Val84 to Leu or Ile76 to Ala (Lemmon et al., 1992Go)—these are precisely the kind of changes observed in the external correlated mutations (see Tables III–VGoGoGo). Similarly, Hebert was able to show that mutation of Gly276, Gly280 and Leu284 to Ala in the helix 6 peptide had a major effect on its ability to inhibit dimerization (Hebert et al., 1996Go). These changes are relatively minor and yet important—we should also note that Gly276 is correlated amongst the adrenergic receptors while Gly280 is conserved amongst the adrenergic receptors. In general, site-directed mutagenesis studies on external residues are limited. However, Huang did observe that mutation of Tyr205 in the neurokinin NK-1 receptor and Tyr206 in the neurokinin NK-2 receptor resulted in loss of activity; the biophysical and mutational data identify this residue as external (Huang et al., 1994Go). Mutation studies were also performed on Phe218 and Phe222 on helix 5 of the {delta}-opioid receptor, along with other aromatic residues, namely Tyr129, Trp173, Trp274 and Tyr308, which were proposed to form a cluster within the transmembrane domain (Befort et al., 1996Go). Neither the F218A mutation nor the F222A mutation had a dramatic affect on binding, but the effect was more marked for the F222A mutation than for the F218A mutation. The authors concluded that Phe222 was probably nearer to the binding site than Phe218 (which is homologous to Ser204 in the ß2-adrenergic receptor). However, their model was based on a bacteriorhodopsin template while our rhodpsin crystal structure-based model suggests that Phe222 is external like its corresponding neurokinin NK-1 residue Tyr205. Thus, one explanation of the reduction in binding affinity of the F222A mutant could reside in reduced stability of the dimer (Phe222 is conserved in the amine and opioid receptors) and this would be compatible with one of the authors conclusions, i.e. that the effect of the mutations on binding could be indirect (see also Table IIIGo) (Gkoutos et al., 1999Go).

Higher order oligomers

Therefore, there is evidence for the mechanisms of dimer formation given in Figure 5Go and the presence of external conserved and external correlated residues on helices 5 and 6 and also on helices 1 and 7 is consistent with this. There is less evidence for the presence of functionality amongst the external residues of helices 2, 3 and 4. [Data on mutation of external residues on helices 2 and 3 is tabulated and briefly discussed elsewhere (Dean et al., 2000; Gouldson et al., 2000Go).] Our current understanding suggests that helix 4 plays only a minor role in GPCR function and is not usually implicated in ligand binding. Even though helix 4 contains few conserved residues even at the sub-family level (such as amongst the ß-adrenergic receptors), it does contain many external correlated mutations. The correlated residues may provide a convenient focus for initiating speculative site-directed mutagenesis studies, particularly in the light of recent evidence to show that helix 4 in rhodopsin is involved in a significant conformational change on activation (Borhan et al., 2000Go; Bourne and Meng, 2000Go).

If helices 2, 3 and 4 do not play a role in dimerization, then they may be involved in higher order oligomerization (Wreggett and Wells, 1995Go; Chidiac et al., 1997Go; Zawarynski et al., 1998Go; Gkoutos et al., 1999Go; Zeng and Wess, 1999Go; George et al., 2000Go; Gouldson et al., 2000Go). Dimerization may be viewed as one step on this pathway towards oligomerization, which in some cases may lead to internalization.

Heterodimerization

Heterodimerization has now been observed for the adenosine (Ciruela et al., 2001Go), angiotensin, bradykinin (AbdAlla et al., 2000Go), chemokine (Mellado et al., 2001Go), dopamine (Rocheville et al., 2000aGo), GABAB (Jones et al., 1998Go; Kaupmann et al., 1998Go; White et al., 1998Go), glutamate (Ciruela et al., 2001Go), muscarinic (Maggio et al., 1999Go; Sawyer and Ehlert 1999Go), opioid (Jordan and Devi, 1999Go), serotonin (Xie et al., 1999Go) and somatostatin receptors (Rocheville et al., 2000aGo,bGo). Based on the occurrence of native-like interactions in the dimer, receptor homodimers may be domain swapped (Figure 5aGo) or contact (Figure 5cGo), while the symmetric chimeric heterodimers (Maggio et al., 1993Go), as shown in Figure 5bGo, are likely to contain only domain swapped dimers. However, receptor heterodimers are likely to contain only contact dimers. The small but growing body of evidence on heterodimerization shows that some subtypes will heterodimerize while others will not. Thus, somatostatin subtype 5 will heterodimerize with subtype 1 but not with subtype 4 (Rocheville et al., 2000bGo). Correlated mutations for external positions in helices 5 and 6 where there is a difference between subtypes 1 and 4 are shown in Table IIIGo. Each of these residues, apart from universal number 515 lie on a common face spanning helices 5 and 6; their identification may help to guide experiments to determine the molecular basis of subtype specificity in GCPR heterodimerization. Table IIIGo also shows that mutation of residues at these positions in other receptors has a distinct effect on function.

For the opioid receptor, subtype specific heterodimerization or heteroligomerization has been reported for {delta}–µ, {delta}{kappa} but not {kappa}–µ (Jordan and Devi, 1999Go; George et al., 2000Go; Gomes et al., 2000Go; Jordan et al., 2000Go,2001Go), and this can be most readily explained by correlated mutations containing different residues for the {delta}, {kappa} and µ subtypes. However, there are only two such correlated mutations (at universal numbering positions 507 and 519) on the external face of helices 5 and 6. For the opioid receptors, it is therefore possible that the molecular basis of the subtype specific heterodimerization resides in higher order combinations of correlated mutations, such as those given in Table IVGo. Pairs of such mutations taken from different networks would give different residue combinations for the {delta}, {kappa} and µ subtypes. Each of the residues in Table IVGo, apart from universal numbers 507 and 606, lie on a common face spanning helices 5 and 6 and have at least one residue from a different network lying nearby. However, if oligomers are involved then the key interface between different subtypes may possibly be the 2,3-interface (Gkoutos et al., 1999Go; Gouldson et al., 2000Go) rather than the 5,6-interface and the relevant correlated residues may be obtained from the web. The role of the second receptor type in modifying the pharmacology would then become analogous to the role of RAMPs (McLatchie et al., 1998Go; Tilakaratne et al., 2000Go; Zumpe et al., 2000Go), which may also bind in the vicinity of helices 1/2 (Gouldson et al., 2000Go; Sexton,P.M., personal communication, 2001).

The heterodimerization data for the chemokine receptors is currently similar to that for somatostatin in that the CCR2 subtype will heterodimerize with CCR5 but not CXCR4 (Mellado et al., 2001Go) and so the molecular basis of specificity most probably resides in external correlated residues on helices 5 and 6 that differ between the CXCR4 and CCR5 subtypes. These residues are listed in Table VGo. However, there is a V64I mutant form of the CXCR4 receptor that does heterodimerize with the CCR2 receptor. Residue 64 is on the external face of helix 1 and points towards helix 2. It may therefore affect binding to the 2,3-interface (Gkoutos et al., 1999Go) so the correlated residues on helix 2 are also listed (the external correlated residues on helix 3 reside between helices 3 and 5).

Thus, while the evolutionary trace method (ET) presented elsewhere (Gkoutos et al., 1999Go) provides a clearer overall mapping of the possible dimerization interfaces (in that the residues identified form readily identifiable clusters on helices 5 and 6), ET analysis of the peptide family fails to identify any residues that differ between the key somatostatin and opioid subtypes. Thus, CMA, when used in conjunction with ET, is likely to provide more precise information on the details of molecular specificity.


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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
A CMA has been performed on the aligned sequences of the neurokinin, opioid, somatostatin, TSH and the whole biogenic amine family of receptors. Through a critical evaluation of models of these receptors based on the rhodopsin crystal structure, we can deduce that a significant proportion of these correlated mutations correspond to external lipid-facing residues. We have shown that correlated and conserved residues are closely linked in several ways. Conserved residues are likely to become correlated residues as one goes back up the dendritic tree, e.g. from the ß2-adrenergic, through the adrenergic, and the catechol receptors to the biogenic amine receptors. Conserved residues are also likely to be flanked by correlated residues since the correlated residues may either be adjacent in the sequence or lie directly above or below the conserved residues in the helical model structures. Definitive proof for a specific functional role for most of the external correlated residues is lacking as not many external residues have been subjected to detailed site-directed mutagenesis experiments. However, Hebert et al. (Hebert et al., 1996Go), Huang et al. (Huang et al., 1994Go) and Befort et al. (Befort et al., 1996Go) have shown that mutation of external residues may affect binding and activity. There is increasing evidence that dimerization plays a role in GPCR activation. It is very likely that the correlated residues are involved in dimer formation, and possibly in the formation of domain swapped dimers (Gouldson and Reynolds, 1997Go; Gouldson et al., 1997bGo,1998Go) as shown in Figure 5Go. The domain swapping hypothesis could readily account for the occurrence of functional residues on the external face of helices 1, 5, 6 and 7. The occurrence of functional residues on the external faces of helices 2, 3 and particularly 4 is a little more puzzling but these residues may be involved in conformational changes or the formation of higher order structures.


    Notes
 
4 To whom correspondence should be addressed. E-mail: c.a.reynolds{at}essex.ac.uk Back


    Acknowledgments
 
We wish to acknowledge the EPSRC (94309861) and the BBSRC (B/06081); we also wish to acknowledge Novo Nordisk for support. The homology, snake diagrams illustrating the position of the correlated mutations and the networks of correlated residues are available from the author's website, http://www.essex.ac.uk/bs/reync/


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Conclusions
 References
 
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Received July 18, 2000; revised June 28, 2001; accepted July 10, 2001.