Structure and function of the ovine type 1 corticotropin releasing factor receptor (CRF1) and a carboxyl-terminal variant.
Corticotropin releasing factor (CRF) is the major neuropeptide regulating the hypothalamo-pituitary-adrenocortical axis in most species. A pituitary receptor for CRF (designated CRF1) belonging to the seven-transmembrane helix, G-protein-coupled receptor superfamily has been cloned for human, rat, mouse and xenopus. Since ovine CRF shares only 84% identity to human/rat CRF (h/rCRF) we postulated that the sheep pituitary CRF1 receptor may have similarly diverged from the rodent and human CRF1. We report the molecular cloning of an ovine pituitary cDNA containing a 1245 bp open reading frame encoding a 415 amino acid sheep CRF1 receptor 78, 86, 94, and 95% homologous to xenopus, chicken, rat, mouse, and human CRF1, respectively. The divergence in primary structure between the sheep CRF1 and the other mammalian CRF1s is primarily localized to the extracellular amino terminal domain of the receptor (18 of 22 divergent residues, ovine vs human CRF1). A variant of the oCRF1 was also isolated (oCRF1var) with 133 bp deleted from nucleotide (nt) 1080 to nt 1213 of the open reading frame (ORF) resulting in a new ORF of 1176 nt predicting a 392 residue CRF1 variant receptor. The 133 bp deletion would cause a frame-shift at residue 358 within the carboxyl-third of the seventh transmembrane domain (TM7) resulting in a shortened cytoplasmic tail with a new amino acid sequence from residue 358 to 392. Scatchard analysis of saturation curves using membrane prepared from Cos 7 cells transfected with oCRF1 or oCRF1var indicated that both wild-type and variant receptors were expressed similarly (number of CRF binding sites) and both bound oCRF with high affinity [oCRF1 (Kd): 2.5 + 1.6 nM; oCRF1var: 5.1 + 2.3 nM]. The non-hydrolyzable GTP analogue (GTPgammaS) lowered the affinity of both wild-type and variant oCRF1 receptors to a similar extent (oCRF1: 18.2 nM; oCRF1var: 22.4 nM). Both wild-type and variant oCRF1 receptors exhibited approximately 10-fold greater selectivity for oCRF and sauvagine compared to h/rCRF or alpha-helical [9-41]oCRF. CRF effectively stimulated the accumulation of cAMP (EC50 = 51 pM) in Cos 7 cells transiently transfected with wild-type but not variant oCRF1 receptor. In Cos 7 cells transfected with oCRF1var, cAMP accumulation was only observed at the highest concentration of oCRF utilized (100 nM). Basal (unstimulated) levels of cAMP in Cos 7 cells transfected with oCRF1var (in the presence of 2 mM IBMX) were approximately 50% lower than for the wild-type oCRF1. Differences in cAMP accumulation could not be attributed to differences in receptor number since total binding sites in the transfected cells were not different between wild-type or variant oCRF1 receptors. Agonist-induced receptor internalization, determined as the percent of total [125I] Tyr0-oCRF bound located in the acid-resistant fraction of transfected Cos 7 cells, increased with time (0-60 min at 37 degrees C) for both wild-type and variant oCRF1. Wild-type CRF1 internalized approximately 2-fold greater percent of total [125I] Tyr0-oCRF bound compared to the variant receptor. In summary, an ovine CRF1 and a CRF1 cytoplasmic tail receptor variant displaying high affinity binding to oCRF as well as selectivity for oCRF vs h/rCRF, were cloned from an adult sheep pituitary cDNA library. GTPgammaS studies indicate that both variant and wild-type receptors couple efficiently to Galphas however, only the wild-type oCRF1 is capable of stimulating cAMP production at physiological levels of CRF. Agonist-induced internalization of the ovine CRF1var is also reduced compared to the wild-type CRF1 receptor. We suggest that the oCRF1var interacts efficiently with Galphas but is unable (post-hormonal binding) to effectively stimulate G-protein activation of adenylate cyclase, indicating that the cytoplasmic tail of the CRF1 can modulate receptor function related to signal transduction. (ABSTRACT TRUNCATED)
Myers
D A
DA
Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA. dean-myers@uokhsc.edu
Trinh
J V
JV
Myers
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D019556:COS Cells; D003001:Cloning, Molecular; D003346:Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D015723:Gene Library; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D010902:Pituitary Gland; D020033:Protein Isoforms / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018019:Receptors, Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012756:Sheep; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D000099052:CRF Receptor, Type 1
D020033:Protein Isoforms; D018019:Receptors, Corticotropin-Releasing Hormone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP