Short N-terminal regions of the calcitonin-like receptor define ligand specificity

Calcitonin (CT), αand β-CT gene-related peptide (CGRP), adrenomedullin(AM), and amylin belong to the calcitonin family of peptides.Six or seven amino acid ring structures, formed by disulfide bonds between cysteine residues, and amidated carboxyl-termini in all these peptides are required for the biological actions.Calcitonin receptors (CTR) and initially orphan CT-like receptors (CLR) with 60% amino acid sequence similarity belong to the class B1 of G proteincoupled receptors (GPCR).The structure of the receptors for CGRP, AM and amylin were established following the discovery in 1998 of three single transmembrane-domain receptor-activity-modifying proteins (RAMP).The RAMP are required as accessory proteins for the functional expression of the CLR.CLR/RAMP1 and -2 heterodimers recognize CGRP and AM, respectively.With RAMP3 the CLR interacts with CGRP and AM.The CTR does not require RAMP to recognize CT, but the receptor associates with RAMP1 and RAMP3 to form mixed type CGRP/amylin and amylin receptors, respectively.In the present thesis, mechanisms of interaction of the CLR with RAMP1 and RAMP2 and their ligands CGRP and AM have been investigated.Eighteen N-terminal extracellular amino acid residues adjacent to the signal sequence of the CLR share little sequence homology with the corresponding region of the CTR.The importance of this sequence for the function of the CLR has been studied.The 18 Nterminal amino acids of the human (h) CLR were either deleted (∆18-hCLR) or substituted by the corresponding sequence of a porcine (p) CTR, to reveal a chimeric pCTR-hCLR.The ∆18-hCLR was inactive in the absence and presence of hRAMP1 or -2.But the pCTR-hCLR coexpressed with hRAMP1 recognized CGRP, while the interaction with AM in the presence of hRAMP2 was abolished.In the mouse (m) CLR, progressive truncation from the N-terminus up to amino acid 20 resulted in a progressive decrease in AM recognition in the presence of mRAMP2.A detailed analysis was achieved with the introduction of deletion and alanine substitution mutations.This allowed us to identify the amino acid sequence 14 to 20 as an important structural domain within the mCLR required for its functional expression as a mCLR/mRAMP2 AM receptor.Importantly, the deletion of the amino acids 14 to 20 of the mCLR minimally affected the CGRP receptor function in the presence of mRAMP1.The substitution of the amino acids 14 to 20 by alanine residues partially restored the functional expression of the mCLR/mRAMP2 AM receptor.Moreover, the deletion of the amino acids 14 to 17 or 14 to 20 in the mCLR altered its structure in a way that chemical cross-linking was no longer possible.