Structure and function of the ovine type 1 corticotropin releasing factor receptor (CRF1) and a carboxyl-terminal variant.
Structure and function of the ovine type 1 corticotropin releasing factor receptor (CRF1) and a carboxyl-terminal variant.
复制标题
绵羊 1 型促肾上腺皮质激素释放因子受体 (CRF1) 和羧基末端变体的结构和功能。
DOI：
10.1016/s0303-7207(98)00157-9
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发表时间：
1998
影响因子：
4.1
通讯作者：
Myers,TR
中科院分区：
文献类型：
--
作者：
Myers,DA;Trinh,JV;Myers,TR
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 CRF1receptor 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 CRF1receptor 78, 86, 94, and 95% homologous to xenopus, chicken, rat, mouse, and human CRF1, respectively. The divergence in primary structure between the sheep CRF1and 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 oCRF1was 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 CRF1variant 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 oCRF1or oCRF1varindicated 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 (GTPγS) lowered the affinity of both wild-type and variant oCRF1receptors to a similar extent (oCRF1: 18.2 nM; oCRF1var: 22.4 nM). Both wild-type and variant oCRF1receptors 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 (EC50=51 pM) in Cos 7 cells transiently transfected with wild-type but not variant oCRF1receptor. 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 ≈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 oCRF1receptors. 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°C) for both wild-type and variant oCRF1. Wild-type CRF1internalized ≈2-fold greater percent of total [125I] Tyr0-oCRF bound compared to the variant receptor. In summary, an ovine CRF1and a CRF1cytoplasmic 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. GTPγS studies indicate that both variant and wild-type receptors couple efficiently to Gαshowever, only the wild-type oCRF1is capable of stimulating cAMP production at physiological levels of CRF. Agonist-induced internalization of the ovine CRF1varis also reduced compared to the wild-type CRF1receptor. We suggest that the oCRF1varinteracts efficiently with Gαsbut is unable …
