Achieving signalling selectivity of ligands for the corticotropin-releasing factor type 1 receptor by modifying the agonist's signalling domain

Background and purpose: Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF1) ligands. Experimental approach: We systematically substituted urocortin, a natural peptide agonist of CRF1, with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF1 to Gs-and G(i)-protein in human embryonic kidney cells, using receptor binding, [S-35]-GTPgS binding stimulation, and cAMP accumulation assays. Key results: Native ligands stimulated G(s) and G(i) activation through CRF1, resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6 -15 led, dependent on the position and nature of the substituent, to ligands that conserved G(s) activity, but were devoid of G(i) activity, only stimulating cAMP accumulation, and competitively antagonized the G(i) activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated G(s) and G(i), as urocortin did. Conclusions and implications: Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF1. This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.