title: The alternatively spliced type II corticotropin-releasing factor receptor, stably expressed in LLCPK-1 cells, is not well coupled to the G protein(s).
abstract: Two alternatively spliced corticotropin-releasing factor receptor (CRF-R) cDNAs, type I and type II, were recently isolated from a human cDNA library. The two cDNAs are identical except that the type II cDNA encodes an additional 29 amino acid inserted in the first putative cytoplasmic loop. Since the first cytoplasmic loop is highly conserved in all the members of the hCRF receptor family we have examined whether the presence of the 29 amino acid cassette in CRF-RII influences G protein coupling in LLCPK-1 cells stably expressing the type I and type II hCRF receptors. Whether measured in intact cells or in membrane preparations, LLCPK-1 cells stably expressing CRF-RII have a 4-5 fold lower binding affinity. Maximal CRF-stimulated cAMP accumulation in LLCPK-1 cells stably expressing CRF-RI was 10-15-fold higher than that in LLCPK-1 cells expressing CRF-RII. The EC50 for CRF-stimulated cAMP accumulation in hCRF-RI-expressing cells was in the range of 0.5 +/- 0.2 nM. In contrast, the EC50 for CRF-stimulated cAMP accumulation in hCRF-RII expressing cells was 7.7 +/- 0.2 nM. hCRF increased phosphoinositide turnover in LLCPK-1 cells stably expressing CRF-RI but not in those expressing CRF-RII; this effect required hCRF concentrations of 100 nM and higher. In membrane preparations, GTP-gamma-S inhibited hCRF binding to CRF-RI and shifted the binding Kd from 4.5 nM to 16.7 nM. Conversely, GTP-gamma-S did not influence hCRF binding to CRF-RII in broken cell membranes. Additionally, CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RI was potentiated by GTP, whereas CRF-stimulated adenylate cyclase activity in cell membranes expressing CRF-RII was insensitive to GTP. These data indicate that CRF-RII is not well coupled to the G protein. Since the only difference between the CRF-RII and CRF-RI is the insert in the first putative cytoplasmic loop, these data indicate that the first cytoplasmic loop plays a crucial role in hCRF receptor coupling to the G protein.
authors.affiliation: Endocrine Unit, Massachusetts General Hospital, Boston 02114, USA.
authors.forename: C
authors.initials: C
authors.lastname: Nabhan
authors.forename: Y
authors.initials: Y
authors.lastname: Xiong
authors.forename: L Y
authors.initials: LY
authors.lastname: Xie
authors.forename: A B
authors.initials: AB
authors.lastname: Abou-Samra
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D018374:LLC-PK1 Cells; D018019:Receptors, Corticotropin-Releasing Hormone / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013552:Swine
chemical_list: D018076:DNA, Complementary; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D019204:GTP-Binding Proteins