14-3-3 structure, function and role in neurodegenerative diseases, trafficking and nuclear signalling

Abstract

Calcitonin gene-related peptide (CGRP) is a 37 amino acid vasodilatory peptide, of which two isoforms, a -C G R P and p-CGRP are known. The use of C-terminal fragments of CGRP, such as C G R P (8-37)Jcd to a pharmacological subdivision of C G R P receptors into CGRP-1 and CGRP-2. We have recently developed the non-peptidc C G R P antagonist, BIBN4096BS, which has sub-nanomolar affinity for primate C G R P receptors. Furthermore, BlBN4096BS is a potent blocker (pKB >lo) of CGRP-induced CAMP stimulation in SK-N-M C cells, human brain microvascular and astroglial cells, CGRP-induced vasodilatation in human temporal arteries, and blocks ncurogenic vasodilatation in the marmoset. BIBN4096 also discriminates between the actions of a -C G R P and [Cys(Et)2,7]hCGRPa in some rat tissues, providing evidence for additional functional C G R P receptor heterogeneity.To further exploit BlBN4096BS as a pharmacological tool, we used BIBN4096BS in porcine coronary vcsscls and cerebral basilar arteries and compared functional with molecular data on C G R P receptor componcnts.The pKB values far BIBN4096BS against [Cys(Et)2,7]hCGRPa, and a -C G R P did not differ significantly (pkB-8), whilst vasodilation of p-CGRP was blocked less potently (pKB-7).Thc analysis of molecular components of the C G R P receptor demonstrated the presence o f hRAMPl and 2, hCRLR and hRCP mRNA suggesting the presence of C G R P l and ADM receptors. O u r data suggest that apart from a isubdivisioni in CGRP-1 and -2 receptors, BIBN4096BS reveals additional functional differences between the actions of a -C G R P and p-CGRP. However, evidence for C G R P receptor heterogeneity on a molecular lcvcl is scarce.RAMPs arc proteins that affect the expression and/or phenotype of the calcitonin receptor (CTR) o r CTR-like receptor (CRLR). I n this study we screened a range of class I1 G protein-coupled receptors (PTHI, PTH2, G H R H , VPAC1, VPACZ receptors) for possible RAMP interactions by measurement of receptor-induced translocation of epitope-tagged RAMPs. Of these, only the VPACl receptor caused significant translocation of all 3 RAMPs, however the PTH2 receptor also translocated RAMP3 to the cell surface. Unlike CTR and CRLR, co-transfcction of VPACl and RAMPs did not alter 1251-VIP (vasoactive intestinal polypeptide) binding and specificity. Furthermore, in contrast to CTR-RAMP interaction, where there was an incrcase in CAMP accumulation and a decrease in phosphoinositol hydrolysis, there was a specific increase in agonist-mediated phosphoinositol hydrolysis when the VPACl receptors were cotransfected with RAMP2. This change was due to an enhancement of Emax with no change in the EC50 value for VIP. N o significant change in CAMP accumulation was observed. This is the first demonstration of an interaction of RAMPs with G protein-coupled receptors outside the C T R family and may suggest a more generalized role for RAMPs in modulating G protein-coupled receptor signalling.The sequencing of human genome is essentially complete and we know its complement of ...