title: Conformational states of the corticotropin releasing factor 1 (CRF1) receptor: detection, and pharmacological evaluation by peptide ligands.
abstract: Previous corticotropin releasing factor 1 (CRF1) receptor characterization has been performed using radiolabeled agonists, which bind predominantly the receptor-G-protein complex. The pharmacological profile of other receptor states, and their abundance, remain poorly characterized. Here we investigated the affinity states of the CRF1 receptor heterologously expressed in Ltk- cells and endogenously expressed in rat cerebellum. In L-CRF1 cell membranes, three agonist affinity states were detected: a very-high affinity receptor-G-protein complex state (eliminated by GTPgammaS) bound by [125I]sauvagine (43 pM, RG); a high affinity state insensitive to GTPgammaS bound by [125I]sauvagine (1.4 nM, termed RO); and a low affinity G-protein-uncoupled state detected by sauvagine displacement of [125I]astressin, a labeled antagonist (120 nM, R). The relative abundance of RG:RO:R was 18%:16%:66%. All three states were demonstrated in rat cerebellum with similar relative abundance (15%:16%:69%). The R state bound CRF with low affinity (270-330 nM), displayed a novel rank order of ligand affinity, and represented the majority of the receptor population in both receptor preparations. This study provides a framework to identify CRF1 receptor conformational states in various receptor preparations.
authors.affiliation: Department of Pharmacology, Neurocrine Biosciences Inc., 10555 Science Center Dr, San Diego, CA 92121-1102, USA. shoare@neurocrine.com
authors.forename: Sam R J
authors.initials: SR
authors.lastname: Hoare
authors.forename: Susan K
authors.initials: SK
authors.lastname: Sullivan
authors.forename: Anil
authors.initials: A
authors.lastname: Pahuja
authors.forename: Nicholas
authors.initials: N
authors.lastname: Ling
authors.forename: Paul D
authors.initials: PD
authors.lastname: Crowe
authors.forename: Dimitri E
authors.initials: DE
authors.lastname: Grigoriadis
mesh_terms: D000595:Amino Acid Sequence; D029845:Amphibian Proteins; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism; D003346:Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D008024:Ligands; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000378:metabolism; D036361:Peptide Hormones; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology*; D011487:Protein Conformation; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000099052:CRF Receptor, Type 1
chemical_list: D029845:Amphibian Proteins; D007457:Iodine Radioisotopes; D008024:Ligands; D010446:Peptide Fragments; D036361:Peptide Hormones; D010455:Peptides; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; C104416:astressin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000099052:CRF Receptor, Type 1; C025727:sauvagine; D003346:Corticotropin-Releasing Hormone