Several receptors which bind the hormone AMY (amylin) with high affinity have now been identified. The minimum binding unit is composed of the CT (calcitonin) receptor at its core, plus a RAMP (receptor activity modifying protein). The receptors have been named AMY1(a), AMY2(a) and AMY3(a) in accordance with the association of the CT receptor (CT(a)) with RAMP1, RAMP2 and RAMP3 respectively. The challenge is now to determine the localization and pharmacological nature of each of these receptors. Recent attempts to achieve these aims will be briefly discussed.
- calcitonin receptor
- receptor activity modifying protein 1 (RAMP1)
AMY, a 37-amino-acid peptide hormone, shares amino acid and structural similarity with CT (calcitonin), CGRP (CT gene-related peptide) and adrenomedullin. Hence, these peptides are grouped together as the CT peptide family . A common pair of cysteine residues in each peptide contributes an N-terminal ring structure that is the key factor in determining the biological activity. Truncation of the sequence encoding this region renders each peptide an antagonist. AMY, a product of the pancreatic islet, has a range of effects that relate to nutritional status, inhibiting food intake, gastric emptying and post-prandial glucagon secretion as well as opposing the metabolic actions of insulin in skeletal muscles .
The molecular nature of AMY receptors
To our knowledge, there is no report describing the identification of a single molecular entity that encodes an AMY receptor. Attempts to identify AMY receptors from tissues or cells that responded to this peptide were problematic due to consistent identification of CT receptors (for which AMY has low affinity) . With hind-sight, such information is extremely informative given that it is now clear that high-affinity AMY-binding sites are formed by CT receptors . The circumstance under which this occurs is extremely unusual. Unlike most G-protein-coupled receptors, the CT receptor can be modified in such a way that its natural affinity for AMY is increased by several orders of magnitude. The proteins that mediate this effect are known as RAMPs (receptor activity modifying proteins), a family of three single-transmembrane spanning proteins . RAMPs and their receptor partners (of which relatively few have been identified) appear to associate in the endoplasmic reticulum, and are co-trafficked to the cell surface where they form stable complexes that act as receptors for such peptides as AMY, CGRP and adrenomedullin . For a discussion of how RAMPs and their receptors interact, see Conner et al. (this issue). In addition to a shift in AMY affinity, the binding profile of other peptides is also altered when the CT receptor is associated with different RAMPs . Thus RAMPs act as pharmacological switches, modulating the responses of cells to extracellular stimuli.
There are minimally six types of human AMY receptor that can be reconstituted in recombinant expression systems . These arise from interactions between the three RAMPs, the CT receptor and its major splice variant (Table 1). If other splice variants of the CT receptor in various species are also considered, the potential receptor diversity and specificity in its actions that AMY could elicit are quite considerable . Although not strictly considered as AMY receptors, CL–RAMP complexes (where CL stands for CT receptor-like receptor) are also capable of binding high concentrations of AMY and could be pharmacologically, if not physiologically, relevant . Thus with appropriate pharmacological characterization, it is probable that all aspects of AMY behaviour can be accommodated by the receptors in this scheme.
AMY receptor pharmacology
The binding properties of the six main receptors have been characterized in some detail. A general pattern has emerged whereby AMY1 receptors have high affinity for salmon CT, AMY and CGRP and lower affinity for mammalian CTs . For AMY2 and AMY3, a similar pattern is principally observed although the affinity for CGRP is lower. Interestingly, the precise receptor phenotype is dependent on cell type and CT receptor splice variant, particularly for RAMP2-generated AMY receptors . Whereas in HEK-293cells, CT(a) is capable of producing adrenomedullin-sensitive receptors with each RAMP, in COS-7 cells, there is essentially no binding or response to this peptide [4,8]. Such variations between studies will probably reflect inherent variability in endogenous RAMP, receptor and G-protein complement.
The profile of peptide interaction at AMY1 receptors has been found to be similar to that for endogenous AMY receptors in mouse α-TSH cells and nucleus accumbens, where high affinity for CGRP in addition to AMY is a feature [3,9]. Cross-reactivity of CGRP and AMY at each other's binding sites is substantial and some biological effects of AMY may be inhibited by the CGRP receptor antagonist CGRP8–37 . Indeed, this resulted in early speculation that AMY mediated its biological actions through CGRP receptors. As AMY is only a weak agonist of cloned CGRP1 receptors, the results are supportive of the existence of a separate subset of receptors that interact closely with AMY and CGRP. This is probably the AMY1 receptor, with the effects of AMY not mimicked by CGRP mediated by AMY2 or AMY3 receptors.
The relative affinities of antagonists at each of the cloned AMY receptors have not been resolved. Nevertheless, salmon (s) CT8–32 was capable of inhibiting AMY responses at AMY1(a), whereas an equal concentration of CGRP8–37 was ineffective . The relative insensitivity of these receptors to CGRP8–37 has led to speculation that this may be the molecular entity responsible for the putative ‘CGRP2’ receptor . Indeed, CGRP8–37 may be useful in distinguishing the effects of CGRP mediated through CGRP1 (CL/RAMP1) and AMY1 receptors. On the other hand, it is unlikely that there is sufficient separation between the effects of sCT8–32 or AC187 (a sCT8–32 analogue) to distinguish between CT and the subtypes of AMY receptor although this has yet to be tested empirically. The effectiveness of AMY8–37, an antagonist of AMY receptors in tissues, has not been reported.
Signal transduction and receptor regulation
Unsurprisingly, given the relatively recent identification of the molecular nature of AMY receptors, there is very little specific literature on the signal transduction, desensitization or other regulation of cloned AMY receptors. However, as the main signalling unit is probably the CT receptor, many of the properties of this receptor could be extrapolated to AMY receptors. Therefore those AMY receptors formed from CT(a) would be expected to signal through Gs and cAMP (as they do) and Gq. On the other hand, those receptors formed from CT(b) might be expected to have reduced signalling capacity and reduced internalization . For CL/RAMP heterodimers, the associated RAMP subtype does not appear to affect appreciably the regulation of CL in terms of internalization and recycling but there has been no investigation of this type for AMY receptors .
Co-localization of AMY receptor components
RAMPs are abundant with the mRNA of at least one RAMP present in almost every tissue or cell line . CT receptors are similarly abundant . A cursory examination of the literature, attempting straightforward overlap of tissue mRNA expression or binding is unlikely to be sufficiently informative, although it is clear that CT receptors are expressed wherever AMY binding is present . A detailed study at the cellular level is required to ascertain the correlation between individual RAMPs, CT receptor splice variant and AMY phenotype. At present, this information is lacking although there has been some recent progress in this regard. In situ hybridization was used to map the localization of CT(a), CT(b) and each RAMP in the rat area prostrema and subfornical organ, areas where circulating AMY is thought to modulate eating and drinking behaviours. mRNA for either one or both CT receptors was observed, along with RAMPs in regions where AMY also stimulated c-fos gene expression . Although there was insufficient resolution to confirm that the RAMP and receptor mRNAs were expressed in the same cells, it is a step forward that function and each receptor component were examined in the same study. CGRP is also able to excite subfornical organ neurons , consistent with at least an AMY1(a) receptor phenotype in these cells.
Cloning of RAMPs has enabled understanding of the molecular basis of AMY receptors. The diversity arising from multiple RAMPs, multiple CTR isoforms and the contribution of other cellular factors provides much distinct and subtle pharmacology, the full impact of which remains to be elucidated.
Signalling Outwards and Inwards: A Focus Topic at BioScience2004, held at SECC Glasgow, U.K., 18–22 July 2004. Edited by J. Challiss (Leicester, U.K.), A. Harwood (University College London, U.K.), M. Humphries (Manchester, U.K.), C. Isacke (Institute of Cancer Research, London, U.K.), R. Liddington (Burnham Institute, La Jolla, CA, U.S.A.), T. Palmer (Glasgow, U.K.), K. Siddle (Cambridge, U.K.), C. Sutherland (Dundee, U.K.), H. Wallace (Aberdeen, U.K.) and M. Welham (Bath, U.K.).
Abbreviations: AMY, amylin; CT, calcitonin; CGRP, CT gene-related peptide; CL, CT receptor-like receptor; RAMP, receptor activity modifying protein
- © 2004 The Biochemical Society