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- Discussion and conclusions
- Conflict of interest
Calcitonin, a 32 amino-acid peptide involved in bone homoeostasis interacts with the family B (secretin-like) G protein-coupled receptor (GPCR), the calcitonin receptor (Sexton et al., 1999). This receptor forms part of a broader subfamily encompassing amylin, calcitonin gene-related peptide (CGRP) and adrenomedullin receptors (Poyner et al., 2002). While calcitonin selectively binds to its namesake receptor or its splice variants, the other pharmacologically defined receptors are heteromeric and are composed of either the calcitonin receptor or the calcitonin receptor-like receptor (CL) in association with receptor activity-modifying proteins (RAMPs) (Poyner et al., 2002).
Transmembrane helices in GPCRs often contain several bends, most of which occur at proline residues. Such distortions in the helices appear to be functionally important; creating movement in the helix, a necessary step in receptor activation (Gether, 2000; Palczewski et al., 2000). In family A (rhodopsin-like) GPCRs, six transmembrane helix kinks are predicted (Yohannan et al., 2004). The function of many of these kink-forming residues has been investigated. For example, simulations have indicated that the movement of transmembrane helical domain 6 (TM6) upon β2-adrenoceptor activation is the result of a change in the kink angle of the conserved proline (Gether et al., 1997). In the human IP prostanoid receptor, proline to alanine substitution of residues in TM6, in particular, resulted in a loss of receptor function (Stitham et al., 2002).
In contrast, only three kinked helices are predicted in family B GPCRs, whereas there are multiple kinks in MRG8 (family C) receptors (Yohannan et al., 2004). The differences in these kink patterns mirrors the fact that signature sequences such as the DRY motif in family A are not found in family B receptors. As such, detailed knowledge of the role of individual residues is required for each receptor family and few assumptions can be made regarding structure-function characteristics. At the present time, rhodopsin is the only GPCR template for modelling and thus ascertaining the true function of residues predicted to be important for structural features in other families of receptor is crucial for interpreting and refining models of them based on this structure.
Proline residues in the transmembrane domains of the two family B GPCRs that have been studied were shown to profoundly influence receptor function. In the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (VPAC) 1 receptor, alanine substitution of each individual proline residue resulted in a marked reduction in Bmax with only small changes in VIP affinity; receptor function was either enhanced or reduced, depending on the position of the mutation (Knudsen et al., 2001). In CL, a component of the CGRP1 receptor, a different pattern was observed; cell surface expression was not affected but the binding and function of two proline to alanine mutants was reduced, most noticeably for P321A in TM6 (Conner et al., 2005).
The human calcitonin receptor shares 55% amino-acid sequence identity with CL and is its closest relative (Figure 1). Consequently, it could be predicted that the conserved transmembrane proline residues in these two receptors would have similar functions. Therefore, in this study, we sought to identify the functional importance of proline residues situated in TM 4, 5 and 6 of the calcitonin receptor, comparing that with earlier data for CL (Conner et al., 2005) in order to help determine whether these residues have family-wide significance, as they do in family A.
Figure 1. Alignment of the insert negative form of the human calcitonin receptor (CT(a)) with its closest relative, the CL showing the conserved transmembrane proline residues in white text on black. Alignment was performed by ClustalW. Predicted signal sequences are italicized, transmembrane (TM) regions are underlined.
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P326A and P336A affected calcitonin receptor function with human but not salmon calcitonin. The data suggest agonist-specific conformations of the human calcitonin receptor, of which those that preferentially bind salmon calcitonin are affected to a lesser degree by the removal of proline than those that bind human calcitonin.
Discussion and conclusions
- Top of page
- Discussion and conclusions
- Conflict of interest
Transmembrane proline residues play a critical role in the fluid movement of GPCR transmembrane helices, a critical step in receptor activation. In family A receptors, the functionality of individual proline residues has been extensively studied. In contrast, in family B GPCRs only the VPAC1 receptor (Knudsen et al., 2001) and more recently, CL (Conner et al., 2005) transmembrane prolines have been studied and therefore it is only an assumption that prolines fulfil a similar role in this GPCR family. Broadly, proline to alanine substitution of some of the residues resulted in altered receptor function, although the precise nature of the alteration was receptor dependent. In this study, we sought to determine the role of the transmembrane proline residues situated in the transmembrane helices of the calcitonin receptor, the closest relative of CL to contribute towards deciphering the function of these residues in family B GPCRs.
Mutation of proline residues at positions 246, 249 and 280 to alanine did not measurably alter receptor function when human calcitonin was used as agonist, consistent with data generated for residues in the same position in CL (Conner et al., 2005). On the other hand, mutation of residues at positions 326 and 336 resulted in reduced function, approximately 165-fold for P326A and 12-fold for P336A. This compares favourably with the loss of function observed with mutation of the equivalent residues in CL, 200-fold for P321A (326 equivalent) and 10-fold for P331A (336 equivalent). Investigation of 125I-hCT membrane binding revealed a different pattern; binding was lost in all mutants. This was unexpected given that function was apparently preserved in three out of five mutants. Membranes used in the binding studies were tested by western blotting and shown to express receptor, indicating that the lack of binding was not due to transfection failure. Therefore, binding in whole cells was investigated in case the membrane preparation process resulted in an artefactual loss of human calcitonin binding at mutant receptors. Indeed, this appeared to be the case as binding could be detected at most of the mutant receptors under whole-cell binding conditions albeit at lower levels than wild-type receptors. On the other hand, binding was still not detectable for P326A, the mutant that had the greatest loss of functionality in response to human calcitonin. There was no reduction in binding affinity for the remaining mutants, although there was an apparent 10-fold increase in affinity at P280A.
Investigation of receptor expression by whole-cell ELISA indicated that P326A and P336A were present at the cell surface at equivalent levels to wild-type receptors. For these mutants, a reduction in human calcitonin interactions did not appear to be due to a change in cell surface expression. In contrast, P246A, P249A and P280A expression was reduced by about half, although this did not reach significance for P249A. Here, there was a reduction in Bmax and expression without any change in potency. Changes in Bmax for these mutants might be related, at least in part, to reduced expression levels.
A point of note is that there appeared to be considerable receptor reserve in our system. Only relatively few receptors were required to elicit a full response; in many cases, Bmax and expression were reduced but functional response maintained. For example, in expression studies, P246A levels were reduced by about half. However, when the DNA concentration was halved, full activity in response to human calcitonin was retained. In our receptor expression (ELISA) assay, a twofold reduction in calcitonin receptor DNA halves the detected signal (data not shown). Together, this suggests that half the receptors can be lost without affecting potency or maximum response.
The extracellular loops and N-terminal domain of the calcitonin receptor are thought to be the primary site of interaction for peptide ligands in common with other family B GPCRs (Dong et al., 2004; Pham et al., 2004, 2005; Hoare, 2005). In this study, we observed that single proline to alanine substitutions in the transmembrane domains of the calcitonin receptor resulted in a reduction in human calcitonin binding. In the VPAC1 receptor, it was reported that binding was not affected by substitution of transmembrane proline residues with alanine. However, the assay used to detect receptor expression was, in fact, a competition binding assay and while it is apparent that binding affinity did not change, the amount of binding detected was reduced in all mutants (Knudsen et al., 2001). When DNA levels were increased, expression (Bmax) returned to wild-type levels. Thus, agonist binding probably was affected in this study and is consistent with the data presented here. At CL, agonist (CGRP)-binding affinity was only substantially reduced by mutation of the residue, which is equivalent to P326 in the calcitonin receptor (Conner et al., 2005). There were no significant changes in Bmax. Interestingly, a truncated form of CGRP, which is an antagonist (CGRP8–37) bound normally suggesting that the proline residue in this position was important for the integrity of a receptor conformation, which binds agonists. We did not have access to an antagonist radioligand to test this for the calcitonin receptor but in terms of reduced human calcitonin receptor-stimulated cAMP activity, P326 in the calcitonin receptor may have a similar function to the equivalent residues in CL. Thus, certain proline residues in the transmembrane domains of the calcitonin receptor may be critical for the appropriate formation of receptor conformations that bind agonists, whether agonist binding is best explained by conformational selection or induced fit. It is worth noting that a two-step-binding mechanism has been proposed for the interaction of peptide ligands with family B GPCRs (Castro et al., 2005; Hoare, 2005). Reduced flexibility in the receptor structure, owing to the loss of proline, is likely to impair this process and hence agonist interactions. Whatever the mechanism of agonist binding, it is likely that the structural change induced by removal of individual prolines modifies either the flexibility or inherent shape of TM6 such that its relationship with the other helices and therefore extracellular loops modifies the interactions of human calcitonin. It is known that changes to the extracellular loops of calcitonin receptors can change binding kinetics. For example, the rat C1b receptor has a 37 amino acid insert in the first extracellular loop; this insert changes the kinetics of 125I-sCT binding, resulting in improved dissociation of the radioligand compared with the rat C1a receptor, which does not have this insert (Houssami et al., 1994). We speculate that a subtle change in the orientation of TM6 could have a secondary effect on the positioning of the other receptor helices leading to modified juxtamembrane and loop presentation to a ligand, thus modulating its interactions.
A second agonist of calcitonin receptors, salmon calcitonin was also tested. In contrast to the human calcitonin cAMP data, receptor function was preserved in all mutants. In CL, there was no apparent difference in the behaviour of proline mutants with agonist; both CGRP and adrenomedullin behaved similarly at each mutant (Conner et al., 2005). Unlike 125I-hCT binding, 125I-sCT membrane binding was detectable in all mutants, consistent with the cAMP data for this agonist. Interestingly, Bmax values were reduced for all mutants apart from P326A. This may be due to decreased expression for P246A, P280A and possibly P249A. Reductions in Bmax for P246A, P249A and P280A did not result in any change in salmon calcitonin affinity or potency, consistent with data for these mutants when human calcitonin was used. On the other hand, there were differences in Bmax for P326A and P336A for the two agonists used. This was most noticeable for P326A where there was no significant reduction in Bmax, affinity or potency for salmon calcitonin but no Bmax could be obtained for human calcitonin and potency was significantly affected by this mutation.
Salmon calcitonin is known to bind essentially irreversibly to calcitonin receptors (Hilton et al., 2000) and this causes persistent activation of cAMP (Michelangeli et al., 1983). Such an avid interaction with the receptors could mask the effect of the proline to alanine substitutions. While salmon and human calcitonin appear to have broadly similar modes of interaction with calcitonin receptors (Dong et al., 2004; Pham et al., 2004), it is conceivable that subtle changes in receptor conformation as might be expected by the removal of proline residues could affect the mode of interaction of one agonist more than the other. Given that salmon calcitonin binds with higher affinity than human calcitonin, the effect of the proline to alanine substitutions on receptor structure may have resulted in greater propensity toward disrupting human calcitonin over salmon calcitonin binding. Interestingly, small modifications to parathyroid hormone (PTH)-related protein, an agonist of the human PTH type 1 receptor, appear to result in distinct conformations of the receptor, based around changes to the movement of TM 5 and 6 (Bisello et al., 2002). Thus, amino acid differences between human and salmon calcitonin could be sufficient to induce distinct calcitonin receptor conformations. There is already some evidence for this in that Gαs supplementation in human embryonic kidney293 cells transfected with the calcitonin receptor increased human calcitonin potency, without altering salmon calcitonin potency (Watson et al., 2000).
The closest relative to the calcitonin receptor is CL and as shown in Figure 1 both of these receptors share the same set of proline residues. However, CL requires RAMPs for function. In order to more accurately compare the consequences of proline to alanine substitution between these two receptors, we transfected our calcitonin receptor proline mutants with RAMP1 to generate AMY1(a) receptors. When stimulated with rat amylin, the resulting changes in agonist potency mirrored those observed with human calcitonin at the calcitonin receptor alone. There were no significant changes with P246A, P249A and P280A but ∼50- and ∼5-fold reductions in amylin potency were observed for P326A and P336A, respectively.
Overall, the effects of the proline to alanine substitutions in the calcitonin receptor appear to be intermediate between effects at CL and the VPAC1 receptors. Human calcitonin potency was reduced for the same residues in CL and the calcitonin receptor. In the VPAC1 receptor, the P326A equivalent (P348A) increased agonist potency and there is no P336A equivalent in this receptor. On the other hand, Bmax values were reduced in the mutant calcitonin receptors as they were in the VPAC1 receptor but not in CL. Expression levels were reduced for some calcitonin receptor mutants but there were no changes in expression for CL, although it should be noted that calcitonin receptor expression studies were performed in the absence of RAMP1, whereas CL studies were with RAMP1. The data presented in this study offer an interesting conundrum; even in closely related receptors, which bind the same family of peptides the effects of proline to alanine substitution are not identical. There are three highly conserved transmembrane proline residues in the family B peptide receptors. These are the equivalents to P246A, P280A and P326A in the human calcitonin receptor. Based on the data available so far for the three family B receptors in which these residues have been studied, it is difficult to predict the consequences of mutating these residues in other family members.
Nonetheless, substituting proline residues with alanine in the transmembrane domains of family B GPCRs, in particular at the P326A equivalent does appear to modify agonist interactions. The data may be consistent with modifications to the movements of TM6, the consequences of which appear to differ between receptors. In the case of the calcitonin receptor, it is apparent that if the agonist has sufficient efficacy, as may be the case for salmon calcitonin, it can overcome the effect of the loss of proline. It is not clear whether the changes to the receptor predominantly affect binding or activation mechanisms. Salmon calcitonin could activate the calcitonin receptor in a different way to human calcitonin and consequently its interactions with the receptor were not affected by the loss of proline. On the other hand, structural modification that affects binding rather than activation could equally well explain the data. The two effects may not be independent of one another and this will require further experimentation to delineate.
In summary, proline residues in the transmembrane helices of family B GPCRs appear to be important for the function of these receptors. However, there is neither sufficient data nor consistency to support a common role for these residues between family A and family B GPCRs. Together, the studies on these residues illustrate that different GPCR families do not necessarily share identical modes of activation and data derived for family A is not necessarily transferable to family B.