REV IEW 0. Poynar. Lecturer, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Depanment of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Pharmaceutical and Blalag!cal Sciences. Aston Uwers~ty. Aston Triangle. Btrmtngham. UK 84 7ET 424 TiPS - December 1995 (Vol. 16) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Pharmacology of receptors for calcitonin gene-related peptide and amylin zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA David Poyner Calcitonin gene-related peptide (CGRP), a widespread neuropeptide with multiple actions, has substantial homology with amylin, a peptide implicated in insulin- resistant diabetes, and adrenomedullin, a recently discovered potent vasodilator. There is controversy over the existence of CGRP receptor subtypes, and whether independent receptors exist for amylin and adrenomedullin. In this article, the current status of CGRP receptor classification is reviewed by David Poyner, taking particular account of species differences, and evidence is presented supporting the existence of multiple receptors for CGRP, as well as independent binding sites for amylin. Calcitonin gene-related peptide (CGRP), a 37 amino acid neuropeptide first identified in 1982 as an extremely potent vasodilatorl, is found throughout the nervous system. It has cardiovascular effects, pro-inflammatory actions and metabolic effects (reduction of glycogen syn- thesis and an increase in glycolysis) that antagonize those of insulin? in this respect, it and amylin are similas. In view of the spectrum of activity of CGRP, and the result- ing clinical implications, there is considerable interest in developing selective agonists and antagonists. No recep- tors for CGRP have yet been cloned, but these are cer- tainly G protein-coupled receptors and linked to adenyl- ate cyclase (and perhaps to other effecters)*. On the basis of pharmacological evidence, a division into CGRP, and CGRP, subtypes has been proposed4. The field is compli- cated by the ability of amylin and adrenomedullin to mimic certain actions of CGRP, leading to uncertainty as to whether these compounds act through CGRP receptors or different receptors. At least one site, identified by radioligand binding (the so-called C3 site), has broad specificity. Structure of CGRP and homologues To appreciate the pharmacology of CGRP and amylin, it is necessary to consider their structures. CGRP exists in two forms in rats and humans, designated aCGRP and PCGRP, or CGRP-I and CGRP-II (Refs 1, 2, 5) (Fig. 1). The main features of the peptides are a disulphide linkage between CyB and Cys7, a probable region of a-helix between residues 12 and 19, and a C-terminal amide. The structure of CGRP is well conserved between species with 75% identity between salmon and human form+. There is considerable conservation of both sequence and structural features between CGRP and peptides of the amylin family, including the N-terminal disulphide bond, C-terminal amide and adjacent regions. There is rather less homology with members of the adrenomedullin fam- ily7,*.The adrenomedullins are over 50 amino acids long; however, removal of the first 12 amino acids of the rat adrenomedullin (15 for human) does not result in any loss of biological activity, and produces a fragment with a N-terminal disulphide bond linking six amino acids also found in CGRP and amyliny. Furthermore, the peptides also have C-terminal amides, which are essential for full activity”. The weakest homology is found with salmon calcitonin (the calcitonin form that is most potent at cal- citonin receptors). This is only 32 residues long; however, this retains a N-terminal disulphide, albeit linking seven amino acids, and a C-terminal amide. Together, CGRP, amylin, adrenomedullin and salmon calcitonin share features that indicate that cross-reactions can occur at common receptors. A major challenge is to discover the preferred receptors for each peptide. CGRP pharmacology A common view of CGRP receptor classification is based on the observation that C-terminal fragments of the peptide, particularly CGRP,,, can act as antagonistsJJ”sl’ (Table 1). These fragments readily antagonize the effects of CGRP on guinea-pig atria, but were much less effective on rat vas deferens. Conversely, the linear CGRP ana- logue [Cys(acetomethoxy)WGRP was a much more potent agonist on rat vas deferens than guinea-pig atriallmll. This led to the proposal that CGRP receptors should be subdivided into CGRP, and CGRP, forms. The C3 site, identified only in binding studies’“, will be discussed later. The CGRP receptor subtype classification has met with some success in accommodating subsequent observations in the field of CGRI’ pharmacology. The CGRP receptor on guinea-pig ileum has been shown to resemble that on atriais. CGRP,,, can also discriminate between receptors found in porcine large- and small-diameter coronary arteriesrh. However, a number of problems have also become apparent. It has never been possible to demon- strate a difference in CGRP,,, affinity in radioligand binding studies between rat vas deferens and other tis- suesisJ7. A number of studies have shown PA, values for CGRP,_,7 in excess of 8.0 (Refs 2,16,18,19), well out of the range of those reported for guinea-pig atria. Conversely, the difference in CGRP,,, affinity between guinea-pig atria and rat vas deferens has been made less clear by reports of pA, values as high as 6.77 for the rat vas defer- ens, and as low as 6.98 for the guinea-pig atria’“J’. In one study this effect was accentuated by the peptidase inhibitor thiorphan, which increased the reported pA, value for CGRP,,, from less than 6.0 to a value of 6.6, leading to the authors to conclude that the selectivity of CGRP,,, was due, at least in part, to an unequal distri- bution of peptidases2’. The apparent selectivity of 0 1995, Elsevier Science Ltd