Structural determinants for binding to CGRP receptors expressed by human SK-N-MC and Col 29 cells: studies with chimeric and other peptides

Abstract

Structure-activity relationships for the binding of human α-calcitonin gene-related peptide 8–37 (hαCGRP_8–37) have been investigated at the CGRP receptors expressed by human SK-N-MC (neuroblastoma) and Col 29 (colonic epithelia) cells by radioligand binding assays and functional assays (hαCGRP stimulation of adenylate cyclase).
On SK-N-MC cells the potency order was hαCGRP_8–37>hαCGRP_19–37=AC187>rat amylin_8–37> hα[Tyr⁰]-CGRP_28–37 (apparent pKBs of 7.49±0.25, 5.89±0.20, 6.18±0.19, 5.85±0.19 and 5.25±0.07). The SK-N-MC receptor appeared CGRP₁-like.
On Col 29 cells, only hαCGRP_8–37 of the above compounds was able to antagonize the actions of hαCGRP (apparent pKB=6.48±0.28). Its receptor appeared CGRP₂-like.
hα[Ala^11,18]-CGRP_8–37, where the amphipathic nature of the N-terminal α-helix has been reduced, bound to SK-N-MC cells a 100 fold less strongly than hαCGRP_8–37.
On SK-N-MC cells, hαCGRP_8–18, _28–37 (M433) and mastoparan-hαCGRP_28–37 (M432) had apparent pKBs of 6.64±0.16 and 6.42±0.26, suggesting that residues 19–27 play a minor role in binding. The physico-chemical properties of residues 8–18 may be more important than any specific side-chain interactions.
M433 was almost as potent as hαCGRP_8–37 on Col 29 cells (apparent pKB=6.17±0.20). Other antagonists were inactive.

Keywords: Calcitonin gene-related peptide, mastoparan, CGRP₁ receptor, CGRP₂ receptor, CGRP antagonist, chimeric peptide