The role of the 8-18 helix of CGRP _8-37 in mediating high affinity binding to CGRP receptors; coulombic and steric interactions

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 [ ¹²⁵I]-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.