Article date: January 2003
By: Stephen G Howitt, Kalle Kilk, Yang Wang, David M Smith, Ulo Langel, David R Poyner in Volume 138, Issue 2, pages 325-332
The role of individual residues in the 8‐18 helix of CGRP8‐37 in promoting high‐affinity binding to CGRP1 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.
Arg11 and Arg18 were replaced by serines to give [Ser11,18]CGRP8‐37. These bound with pKi values <6 to SK‐N‐MC cells and had apparent pA2 values of 5.81±0.04 and 5.31±0.11 on SK‐N‐MC and L6 cells. CGRP8‐37 had a pKi of 8.22 on SK‐N‐MC cells and pKb values on the above cell lines of 8.95±0.04 and 8.76±0.04.
The arginines were replaced with glutamic acid residues. [Glu11]CGRP8‐37 had a pKb of 7.14±0.14 on SK‐N‐MC cells (pKi=7.05±0.05) and 6.99±0.08 on L6 cells. [Glu18]CGRP8‐37 had a pKb of 7.10±0.0.08 on SK‐N‐MC cells (pKi=6.91±0.23) and 7.12±0.09 on L6 cells.
Leu12, Leu15 and Leu16 were replaced by benzoyl‐phenylalanine (bpa) residues. On SK‐N‐MC cells, the apparent pA2 values of [bpa12]‐, [bpa15]‐ and [bpa16]CGRP8‐37 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).
It is concluded that the Arg11 and Arg18 are involved in specific electrostatic interactions with other residues, either on the CGRP1 receptors or elsewhere on CGRP8‐37. Leu16 is in a conformationally restricted site when CGRP8‐37 binds to CGRP1 receptors, unlike Leu12 and Leu15.
The role of individual residues in the 8‐18 helix of CGRP8‐37 in promoting high‐affinity binding to CGRP1 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.
Arg11 and Arg18 were replaced by serines to give [Ser11,18]CGRP8‐37. These bound with pKi values <6 to SK‐N‐MC cells and had apparent pA2 values of 5.81±0.04 and 5.31±0.11 on SK‐N‐MC and L6 cells. CGRP8‐37 had a pKi of 8.22 on SK‐N‐MC cells and pKb values on the above cell lines of 8.95±0.04 and 8.76±0.04.
The arginines were replaced with glutamic acid residues. [Glu11]CGRP8‐37 had a pKb of 7.14±0.14 on SK‐N‐MC cells (pKi=7.05±0.05) and 6.99±0.08 on L6 cells. [Glu18]CGRP8‐37 had a pKb of 7.10±0.0.08 on SK‐N‐MC cells (pKi=6.91±0.23) and 7.12±0.09 on L6 cells.
Leu12, Leu15 and Leu16 were replaced by benzoyl‐phenylalanine (bpa) residues. On SK‐N‐MC cells, the apparent pA2 values of [bpa12]‐, [bpa15]‐ and [bpa16]CGRP8‐37 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).
It is concluded that the Arg11 and Arg18 are involved in specific electrostatic interactions with other residues, either on the CGRP1 receptors or elsewhere on CGRP8‐37. Leu16 is in a conformationally restricted site when CGRP8‐37 binds to CGRP1 receptors, unlike Leu12 and Leu15.
British Journal of Pharmacology (2003) 138, 325–332. doi:10.1038/sj.bjp.0705040
DOI: 10.1038/sj.bjp.0705040
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