From Micromolar to Nanomolar Affinity:  A Systematic Approach To Identify the Binding Site of CGRP at the Human Calcitonin Gene-Related Peptide 1 Receptor

Abstract

CGRP Y0-28-37 is known as a selective CGRP1 receptor antagonist. In order to elucidate the essential requirements for its receptor interaction, we performed a variety of systematic approaches by modifying the C-terminal segments CGRP Y0-28-37 and CGRP 27-37. N-Terminal and C-terminal segments have been synthesized, as well as chimeras which combine segments of CGRP, adrenomedullin, and amylin. Furthermore, we carried out an Ala scan, a Phe scan, a D-amino acid scan and a Pro scan of CGRP 27-37. Additionally, single amino acids were replaced by those with similar biophysical properties. Receptor binding studies of all analogs were performed at human neuroblastoma cells SK-N-MC, which selectively express the hCGRP1 receptor. On the basis of the obtained results, we synthesized a series of ligands with multiple amino acid replacements in order to optimize the exchange at each position. This approach yielded to a series of high affinity ligands, including [D31,P34,F35] CGRP 27-37 which exhibits a 100-fold increased affinity compared to the unmodified segment. So far, this is the smallest CGRP analog that shows affinity in the nanomolar range.

Selective CGRP and adrenomedullin peptide binding by tethered RAMP‐calcitonin receptor‐like receptor extracellular domain fusion proteins

“…We used the tethered fusion proteins to identify critical determinants of CGRP and AM binding to their receptor ECD complexes. Consistent with previous studies, 22,26,37 we observed that the minimal CGRP(27-37)NH 2 fragment retained essentially wild-type binding. Our ALA-scan experiments were in agreement with those of other groups 22,26 that CGRP residues T30, V32, and F37 are most critical for ECD binding.…”

“…Previous studies indicated that the C-terminal (27-37) fragment of CGRP retained the ability to bind the CGRP receptor, albeit with low affinity. 22,26,36,37 It was recently demonstrated that the C-terminal eight amino acids of AM, in the context of the 22-52 fragment, constituted the primary AM receptor ECD binding epitope. 27 We examined the ability of N-terminally truncated CGRP and AM peptides of varying lengths to bind their respective tethered receptor ECD fusion proteins in the competition AlphaScreen assay.…”

“…21 It is unclear if CGRP and AM bind to their receptor ECDs as continuous a-helices as observed for other class B GPCR ECD-peptide pairs. Chimeric receptor and mutagenesis studies indicated that the RAMP ECDs are sufficient to determine peptide selectivity and identified receptor and CGRP and AM peptide residues that are important for their interactions, [22][23][24][25][26][27] but precisely how RAMPs determine peptide selectivity remains uncertain. The CGRP and AM receptor stoichiometries are a topic of debate.…”

Monitoring of the internalization of neuropeptide Y on neuroblastoma cell line SK‐N‐MC

“…Each Fmoc-amino acid (10-fold excess) was introduced by double coupling (2 Â 36 min) using in situ activation with diisopropylcarbodiimide and hydroxybenzotriazole. Fmoc removal was carried out with piperidine in dimethylformamide (15 min) [10]. Prior to cleavage of the peptides from the resins, they were N-terminally modified.…”

The First Selective Agonist for the Neuropeptide YY5Receptor Increases Food Intake in Rats

“…One limitation of the agonists currently used for in vivo studies is the lack of receptor selectivity; NPY and PYY bind to the receptors Y 1 (12), but antagonism against NPY-induced increase in food intake has been observed as well (16,17 Because highly specific tools to investigate the Y 5 receptor activity are still missing, we have focused our work on the design of NPY receptor agonists with both high affinity and selectivity for the Y 5 subtype. It is well established that the C-terminal part of NPY represents the interaction site with the Y receptors and that amino acid exchange is poorly tolerated in the region 33-36 (49); therefore, we induced a conformational change within the peptide region that mediates receptor binding by introducing the ␤-turn-inducing dipeptide Ala-Aib (aminoisobutyric acid) (19) (21). Each Fmoc-amino acid (10-fold excess) was introduced by double coupling (twice for 36 min) using in situ activation with diisopropylcarbodiimide and hydroxybenzotriazole.…”