SIGNALING PROPERTIES OF MOUSE AND HUMAN CORTICOTROPIN-RELEASING FACTOR (CRP) RECEPTORS - DECREASED COUPLING EFFICIENCY OF HUMAN TYPE-II CRFRECEPTOR

CRF is the primary neuroregulator of the function of the hypothalamic- pituitary-adrenal axis. We have recently cloned a mouse CRF receptor ( mCRF-R) complementary DNA (cDNA) from an AtT-20 cell cDNA library by p olymerase chain reaction. To compare the functions of mCRF-R to those of the human type I and type II CRF receptors (hCRF-RI and hCRF-RII), cDNAs were cloned into the expression vector pcDNA1 and transfected in to COS-7 cells. CRF binding and CRF-stimulated cAMP accumulation as we ll as phosphoinositide hydrolysis were measured. Scatchard analysis of the binding of I-125-labeled [Tyr(0)]r/hCRF ([I-125]CRF) to, COS-7 ce lls expressing mCRF-R and hCRF-RI cDNAs revealed the same apparent K-d (9 nM). In contrast, the apparent binding K-d for hCRF-RII was 20 nM CRF. Maximal stimulatory concentrations (1 mu M) of rat/human CRF-(1-4 1) (r/hCRF) increased cAMP accumulation in COS-7 cells transfected wit h mCRF-R, hCRF-RI, and hCRF-RII cDNA. plasmid (10 mu g each) from basa l values of 8-19 pmol/10(5) cells . 15 min to 84 +/- 10, 87 +/- 16, an d 45 +/- 16 pmol/10(5) cells . 15 min, respectively The EC(50) values of r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing mCRF- R and hCRF-RI cDNAs were similar at 0.4 +/- 0.2 and 0.7 +/- 0.2 nM, re spectively. Conversely, the EC(50) of r/hCRF-stimulated cAMP accumulat ion in hCRF-RII-transfected COS-7 cells was 47.5 +/- 18.9 nM. As the l evel of expression of hCRF-RII was lower than that of hCRF-RI, we comp ared r/hCRF-stimulated cAMP accumulation in COS-7 cells expressing low and high levels of hCRF-RI. The EC(50) for r/hCRF-stimulated cAMP acc umulation in COS-7 cells transfected with hCRF-RI did not change when receptor expression was varied by a factor of 1- to 8.4-fold. In contr ast, the EC(50) for r/hCRF-stimulated cAMP accumulation mediated by hC RF-RII was at least 100-fold higher than that mediated by the hCRF-RI in COS-7 cells, which suggests poor coupling between hCRF-RII and aden ylate cyclase. Inositol phosphate (IF) levels were also determined in mCRF-R, hCRF-RI, and hCRF-RII cDNA-transfected COS-7 cells stimulated with increasing concentrations of r/hCRF. r/hCRF-stimulated IPs accumu lation was dose dependent in COS-7 cells expressing mCRF-R and hCRF-RI using 100 and 1000 nM r/hCRF. Concentrations of 10 (or less) nM r/hCR F had no effect on IP generation. hCRF-RII did not mediate stimulation of IP even at 1000 nar r/hCRF. These data indicate that mCRF-R and hC RF-RI have similar binding and signaling properties. In contrast, hCRF -RII did not increase phosphoinositide tw?lover. Additionally, hCRF-RI I had a 1.8-fold lower expression level, 2.1-fold lower apparent bindi ng affinity, and at least 100-fold lower coupling to stimulation of cA MP accumulation in COS-7 cells.