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
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Abstract |
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Keywords: dimerization/G-protein/lipid-facing/mutations/rhodopsin crystal structure
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Introduction |
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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., 1997) 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, 1994
; Herzyk and Hubbard, 1995
); in other families (e.g. neurokinin, glucagon) the N-terminus and the extracellular loops are also involved in ligand binding (Fong et al., 1993
; Buggy et al., 1995
) while in others (e.g. metabotropic glutamate) the ligand binds to the N-terminus (Kunishima et al., 2000
). 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., 1997
). 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., 1994; Singer et al., 1995
; Pazos et al., 1997
). The technique was greeted with much enthusiasm as a way of predicting residueresidue contacts within a protein core and therefore for predicting three-dimensional structure from sequence (Gobel et al., 1994
; Benner, 1995
; Ortiz et al., 1998
). In many protein families this potential has not been fully realized (Taylor and Hatrick, 1994
) but in the GPCRs, the technique has yielded interesting observations (Kuipers et al., 1997
; Horn et al., 1998a
), 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., 1998b
; Vriend, 2000
), the receptors have appropriately developed dendritic trees and model structures can be generated by homology (Higgs and Reynolds, 2000
) to the rhodopsin crystal structure (Palczewski et al., 2000
; Teller et al., 2001
) to assist in the interpretation of the results. Moreover, recent research (Pazos et al., 1997
; Nilsson et al., 1999
) has supported earlier findings (Oliveira et al., 1993
; Horn et al., 1998a
) 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., 1997a,1998
), as summarized in Figure 1
, 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 proteinprotein 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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Methods |
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Results |
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Discussion |
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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, 1993; Luo et al., 1994
; Farahbakhsh et al., 1995
; Gether et al., 1995
,1997a
, b
; Altenbach et al., 1996
; Javitch et al., 1997
). However, it is clear that most of the correlated residues identified as external must remain so. Analysis of Figure 1
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., 1996
; Gouldson et al., 1997b
; Konvicka et al., 1998
; Higgs and Reynolds, 2000
), 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., 1995; Elling and Schwartz 1996
) and the
-opioid (Thirstrup et al., 1996
) 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
-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 receptorsat 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., 1999
). 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 ß switch (Suryanarayana et al., 1991
; Suryanarayana and Kobilka, 1993
; Gouldson et al., 1997c
). Table II
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
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, 1998
). They may modulate the agonist-induced conformational change through effects on helixhelix 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 Asn312 (ß2-AR numbering) (Gouldson et al., 1997b
,c
). 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 4. 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 II
, 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., 1996
), which in some respects are similar to the correlated residues (Gkoutos et al., 1999
).
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., 1994; Puffenberger et al., 1994
), 515 (Kosugi and Mori, 1996
), 519 (Hwa et al., 1995
), 526 (Ji et al., 1995
) and 616 (Tomic et al., 1993
; Hebert et al., 1996
); 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 helixhelix interactions, either during conformational changes within the monomer or during oligomer formation.
Recent work (Oliveira et al., 1993; Pazos et al., 1997
; Horn et al., 1998a
) 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., 1999
). 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., 1998,2000
); 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., 1996
) 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., 1996
). Immunological studies (Ciruela et al., 1995
) 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., 1993,1996
) provide some of the most compelling evidence for dimerization. Maggio constructed chimeric receptors where the A domain (including helices 15) 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 muscarinicadrenergic 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 5
, 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., 1997
), though domain swapping may require some additional degree of unfolding (Schulz et al., 2000
;Rousseau et al., 2001
). 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., 1996
)except here we conclude that domain swapping resulted in the formation of a 4,5-domain swapped dimer (see Gouldson et al., 1998
) 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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Higher order oligomers
Therefore, there is evidence for the mechanisms of dimer formation given in Figure 5 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., 2000
).] 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., 2000
; Bourne and Meng, 2000
).
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, 1995; Chidiac et al., 1997
; Zawarynski et al., 1998
; Gkoutos et al., 1999
; Zeng and Wess, 1999
; George et al., 2000
; Gouldson et al., 2000
). 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., 2001), angiotensin, bradykinin (AbdAlla et al., 2000
), chemokine (Mellado et al., 2001
), dopamine (Rocheville et al., 2000a
), GABAB (Jones et al., 1998
; Kaupmann et al., 1998
; White et al., 1998
), glutamate (Ciruela et al., 2001
), muscarinic (Maggio et al., 1999
; Sawyer and Ehlert 1999
), opioid (Jordan and Devi, 1999
), serotonin (Xie et al., 1999
) and somatostatin receptors (Rocheville et al., 2000a
,b
). Based on the occurrence of native-like interactions in the dimer, receptor homodimers may be domain swapped (Figure 5a
) or contact (Figure 5c
), while the symmetric chimeric heterodimers (Maggio et al., 1993
), as shown in Figure 5b
, 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., 2000b
). Correlated mutations for external positions in helices 5 and 6 where there is a difference between subtypes 1 and 4 are shown in Table III
. 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 III
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 µ,
but not
µ (Jordan and Devi, 1999
; George et al., 2000
; Gomes et al., 2000
; Jordan et al., 2000
,2001
), and this can be most readily explained by correlated mutations containing different residues for the
,
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 IV
. Pairs of such mutations taken from different networks would give different residue combinations for the
,
and µ subtypes. Each of the residues in Table IV
, 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., 1999
; Gouldson et al., 2000
) 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., 1998
; Tilakaratne et al., 2000
; Zumpe et al., 2000
), which may also bind in the vicinity of helices 1/2 (Gouldson et al., 2000
; 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., 2001) 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 V
. 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., 1999
) 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., 1999) 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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Conclusions |
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Notes |
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Acknowledgments |
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References |
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Received July 18, 2000; revised June 28, 2001; accepted July 10, 2001.