Extracellular loops 1 and 3 and their associated transmembrane regions of the calcitonin receptor-like receptor are needed for CGRP receptor function

Abstract

The first and third extracellular loops (ECL) of G protein-coupled receptors (GPCRs) have been implicated in ligand binding and receptor function. This study describes the results of an alanine/leucine scan of ECLs 1 and 3 and loop-associated transmembrane (TM) domains of the secretin-like GPCR calcitonin receptor-like receptor which associates with receptor activity modifying protein 1 to form the CGRP receptor. Leu195Ala, Val198Ala and Ala199Leu at the top of TM2 all reduced αCGRP-mediated cAMP production and internalization; Leu195Ala and Ala199Leu also reduced αCGRP binding. These residues form a hydrophobic cluster within an area defined as the "minor groove" of rhodopsin-like GPCRs. Within ECL1, Ala203Leu and Ala206Leu influenced the ability of αCGRP to stimulate adenylate cyclase. In TM3, His219Ala, Leu220Ala and Leu222Ala have influences on αCGRP binding and cAMP production; they are likely to indirectly influence the binding site for αCGRP as well as having an involvement in signal transduction. On the exofacial surfaces of TMs 6 and 7, a number of residues were identified that reduced cell surface receptor expression, most noticeably Leu351Ala and Glu357Ala in TM6. The residues may contribute to the RAMP1 binding interface. Ile360Ala impaired αCGRP-mediated cAMP production. Ile360 is predicted to be located close to ECL2 and may facilitate receptor activation. Identification of several crucial functional loci gives further insight into the activation mechanism of this complex receptor system and may aid rational drug design.

Fig. 1

Snake plot of transmembrane domain of CLR. Amino acids of CLR between 125 and 397 are depicted as circles containing 1-letter identifiers. The putative boundaries of the TM regions are shown. Residues that were mutated in the ECL1 and ECL3 regions are shaded.

Fig. 2

Representative dose–response curves of ECL mutants that showed a significant decrease in αCGRP potency for cAMP production. Sigmoidal concentration–response curves comparing the WT receptor and mutant receptors are shown. Each WT and mutant receptor concentration–response comparison curve is a representative example from at least three independent experiments. Each assay point was performed in duplicate where each point on the graph represents the mean ± S.E.M.

Fig. 3

Representative dose–response curves of mutants that showed a significant increase in αCGRP potency. Sigmoidal concentration–response curves comparing the WT receptor and mutant receptors are shown. Each WT and mutant receptor concentration–response comparison curve is a representative example from at least three independent experiments. Each assay point was performed in duplicate where each point on the graph represents the mean ± S.E.M.

Fig. 4

Representative inhibition curves of ECL mutant receptors that significantly alter CGRP binding. Sigmoidal αCGRP inhibition curves comparing the WT receptor and mutant receptors are shown. Each WT and mutant receptor curve is a representative example of at least three independent experiments. Each assay point was performed in duplicate where each point on the graph represents the mean ± S.E.M.

Fig. 5

Important residues affiliated with the TM1–TM2–TM7 region of CLR. a) Side view of the CLR TM domain model generated from bovine rhodopsin (PDB accession code 1U19) showing the relative position of Leu195, Val198 and Ala199. A transparent ribbon represents the TM helical arrangement, where TM1, 5, 6 and 7 have been labeled. ECL1 and associated TM2 is highlighted by an opaque ribbon. b) Extracellular view of the CLR TM model (PDB accession code 1U19) represented by transparent ribbon with depth cue perception. Ile371 is located within TM7. Ala203 and Ala206 are predicted to be in the middle of ECL1. Leu195, Val198 and Ala199 are located at the top of TM2.

Fig. 6

Predicted location of H219, L220, L222 and I360. a) Side view of the CLR TM domain model generated from bovine rhodopsin (PDB accession code 3DQB) represented by transparent ribbon. TM3, ECL3 and ECL2 are opaque ribbons. His219, Leu220 and Leu222 are located within TM3. Ile360 is located within ECL3 in close proximity to ECL2. b) Extracellular view of the CLR TM model. ECL2 is transparent. The same residues as above are shown.

Fig. 7

Putative RAMP1 interface. Side view of the CLR TM domain model generated from bovine rhodopsin (PDB accession code 3DQB) represented by transparent ribbons. TM6 and TM7 highlighted by opaque ribbons. The side chains of Leu351, Arg355, Gly358, Lys359, Glu362, Tyr365, Tyr367 and Ile357 are highlighted to represent residues that were found to reduce cell surface expression and predicted in this model to face outward toward the supposed lipid environment.