The role of the 8-18 helix of CGRP_(8-37) in mediating high affinity binding to CGRP receptors; coulombic and steric interactions

摘要

1 The role of individual residues in the 8-18 helix of CGRP_(8-37) in promoting high-affinity binding to CGRP_1 receptors expressed on rat L6 and human SK-N-MC cells has been examined. The relative potencies of various derivatives were estimated from their ability to inhibit the human αCGRP-mediated increase in cyclic AMP production and the binding of [~(125)I]-human αCGRP. 3 Arg~(11) and Arg~(18) were replaced by serines to give [Ser~(11,18)]CGRP_(8-37). These bound with pKi values <6 to SK-N-MC cells and had apparent pA_2 values of 5.81 +- 0.04 and 5.31 +- 0.11 on SK-N-MC and L6 cells. CGRP_(8-37) had a pKi of 8.22 on SK-N-MC cells and pK_b values on the above cell lines of 8.95 +- 0.04 and 8.76 +- 0.04. 3 The arginines were replaced with glutamic acid residues. [Glu~(11)CGRP_(8-37) had a pK_b of 7.14 +- 0.14 on SK-N-MC cells (pKi= 7.05 +- 0.05) and 6.99 +- 0.08 on L6 cells. [Glu~(18)]CGRP_(8-37) had a pK_b of 7.10 +- 0.0.08 on SK-N-MC cells (pKi= 6.91 +- 0.23) and 7.12 +- 0.09 on L6 cells. 4 Leu~(12), Leu~(15) and Leu~(16) were replaced by benzoyl-phenylalanine (bpa) residues. On SK-N-MC cells, the apparent pA_2 values of [bpa~(12)]-, [bpa~(15)]- and [bpa~(16)]CGRP_(8-37) were respectively 7.43 +- 0.23, 8.34 +- 0.11 and 5.66 +- 0.16 (pKi values of 7.14 +- 0.17, 7.66 +- 0.21 and <6): on L6 cells they were 7.96 +- 0.36, 8.28 +- 0.21 and 6.09 +- 0.04 (all n = 3). 5 It is concluded that the Arg~(11) and Arg~(18) are involved in specific electrostatic interactions with other residues, either on the CGRP_1 receptors or elsewhere on CGRP_(8-37). Leu~(16) is in a conformationally restricted site when CGRP_(8-37) binds to CGRP_1 receptors, unlike Leu~(12) and Leu~(15).