| Popis: |
Introduction Most of the pharmaceuticals today target at G protein-coupled receptors (GPCRs) [1] that transmit extracellular signals into cells. GPCRs are promiscuous, that means a single receptor can activate different signalling events, presumably via different G protein subtypes. Functional selectivity of GPCR ligands has been observed and points at a new orientation in pharmaceutical research. However, the structural characteristics of ligands that produce this selectivity are far from known [2]. The polypeptide hormone urocortin I (Ucn) activates both Gs and Gi proteins via the corticotropin-releasing factor receptor type 1 (CRF1). We have recently developed an easy method for the separate measurement of Gs and Gi activation at HEK 293 cells stably transfected with cDNA coding for CRF1 [3,4]. The aim of this study was to search for structural determinants of the peptide agonist Ucn (DDPPLSIDLT FHLLRTLLEL ARTQSQRERA EQNRIIFDSV-NH2) that direct the signalling to Gs and Gi, respectively. For this purpose, the effect of single replacements by bulky amino acids, benzoyl-phenylalanine (Bpa) and naphthyl alanine (Nal), on Gs and Gi signalling pathways was measured in HEK-CRF1 cells, using receptor binding, [S]-GTPγS binding stimulation, and cAMP accumulation assays. |
