Structure and function of the ovine type 1 corticotropin releasing factor receptor (CRF 1) and a carboxyl-terminal variant

Abstract

Corticotropin releasing factor (CRF) is the major neuropeptide regulating the hypothalamo–pituitary–adrenocortical axis in most species. A pituitary receptor for CRF (designated CRF 1) 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 CRF 1 receptor may have similarly diverged from the rodent and human CRF 1. We report the molecular cloning of an ovine pituitary cDNA containing a 1245 bp open reading frame encoding a 415 amino acid sheep CRF 1 receptor 78, 86, 94, and 95% homologous to xenopus, chicken, rat, mouse, and human CRF 1, respectively. The divergence in primary structure between the sheep CRF 1 and the other mammalian CRF 1s is primarily localized to the extracellular amino terminal domain of the receptor (18 of 22 divergent residues, ovine vs human CRF 1). A variant of the oCRF 1 was also isolated (oCRF 1var) 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 CRF 1 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 oCRF 1 or oCRF 1var indicated that both wild-type and variant receptors were expressed similarly (number of CRF binding sites) and both bound oCRF with high affinity [oCRF 1 ( K d): 2.5+1.6 nM; oCRF 1var: 5.1+2.3 nM]. The non-hydrolyzable GTP analogue (GTP γS) lowered the affinity of both wild-type and variant oCRF 1 receptors to a similar extent (oCRF 1: 18.2 nM; oCRF 1var: 22.4 nM). Both wild-type and variant oCRF 1 receptors exhibited ≈10-fold greater selectivity for oCRF and sauvagine compared to h/rCRF or α-helical [9–41]oCRF. CRF effectively stimulated the accumulation of cAMP (EC 50=51 pM) in Cos 7 cells transiently transfected with wild-type but not variant oCRF 1 receptor. In Cos 7 cells transfected with oCRF 1var, 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 oCRF 1var (in the presence of 2 mM IBMX) were ≈50% lower than for the wild-type oCRF 1. 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 oCRF 1 receptors. Agonist-induced receptor internalization, determined as the percent of total [ 125I] Tyr 0-oCRF bound located in the acid-resistant fraction of transfected Cos 7 cells, increased with time (0–60 min at 37°C) for both wild-type and variant oCRF 1. Wild-type CRF 1 internalized ≈2-fold greater percent of total [ 125I] Tyr 0-oCRF bound compared to the variant receptor. In summary, an ovine CRF 1 and a CRF 1 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. GTP γS studies indicate that both variant and wild-type receptors couple efficiently to G αs however, only the wild-type oCRF 1 is capable of stimulating cAMP production at physiological levels of CRF. Agonist-induced internalization of the ovine CRF 1var is also reduced compared to the wild-type CRF 1 receptor. We suggest that the oCRF 1var interacts efficiently with G αs but is unable (post-hormonal binding) to effectively stimulate G-protein activation of adenylate cyclase, indicating that the cytoplasmic tail of the CRF 1 can modulate receptor function related to signal transduction. The cytoplasmic carboxyl terminus of the CRF 1 also appears to play a role in agonist-induced internalization of this member of the class II subfamily of G-protein coupled receptors (GPCRs).