Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity

Human galanin is a 30 amino acid neuropeptide that elicits arange of biological activities by interaction with G protein-coupledreceptors. We have generated a model of the human GALR1 galaninreceptor subtype (hGALR1) based on the alpha carbon maps offrog rhodopsin and investigated the significance of potentialcontact residues suggested by the model using site-directedmutagenesis. Mutation of Phe186 within the second extracellularloop to Ala resulted in a 6-fold decrease in affinity for galanin,representing a change in free energy consistent with hydrophobicinteraction. Our model suggests interaction between Phe186 ofhGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificitywas investigated by replacement of residues in hGALR1 with thecorresponding residues in hGALR2 and use of the hGALR2-specificligands hGalanin(2–30) and [D-Trp2]hGalanin(1–30).The His267Ile mutant receptor exhibited a pharmacological profilecorresponding to that of hGALR1, suggesting that His267 is notinvolved in a receptor–ligand interaction. The mutationPhe115Ala resulted in a decreased binding affinity for hGalaninand for hGALR2-specific analogues, indicating Phe115 to be ofstructural importance to the ligand binding pocket of hGALR1but not involved in direct ligand interaction. Analysis of Glu271Trpsuggested that Glu271 of hGALR1 interacts with the N-terminusof galanin and that the Trp residue in the corresponding positionin hGALR2 is involved in receptor subtype specificity of binding.Our model supports previous reports of Phe282 of hGALR1 interactingwith Trp2 of galanin and His264 of hGALR1 interacting with Tyr9of galanin.