Nabhan C, Xiong Y, Xie L Y, Abou-Samra A B
Endocrine Unit, Massachusetts General Hospital, Boston 02114, USA.
Biochem Biophys Res Commun. 1995 Jul 26;212(3):1015-21. doi: 10.1006/bbrc.1995.2071.
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.
最近从人cDNA文库中分离出两种选择性剪接的促肾上腺皮质激素释放因子受体（CRF-R）cDNA，即I型和II型。这两种cDNA除了II型cDNA在第一个假定的细胞质环中编码额外的29个氨基酸外是相同的。由于第一个细胞质环在hCRF受体家族的所有成员中高度保守，我们研究了CRF-RII中29个氨基酸盒的存在是否会影响稳定表达I型和II型hCRF受体的LLCPK-1细胞中的G蛋白偶联。无论是在完整细胞还是在膜制剂中进行测量，稳定表达CRF-RII的LLCPK-1细胞的结合亲和力都低4至5倍。在稳定表达CRF-RI的LLCPK-1细胞中，CRF刺激的cAMP积累最大值比表达CRF-RII的LLCPK-1细胞高10至15倍。在表达hCRF-RI的细胞中，CRF刺激的cAMP积累的EC50在0.5±0.2 nM范围内。相比之下，在表达hCRF-RII的细胞中，CRF刺激的cAMP积累的EC50为7.7±0.2 nM。hCRF增加了稳定表达CRF-RI的LLCPK-1细胞中的磷酸肌醇周转率，但在表达CRF-RII的细胞中没有增加；这种效应需要100 nM及更高浓度的hCRF。在膜制剂中，GTP-γ-S抑制hCRF与CRF-RI的结合，并使结合Kd从4.5 nM变为16.7 nM。相反，GTP-γ-S在破碎细胞膜中不影响hCRF与CRF-RII的结合。此外，在表达CRF-RI的细胞膜中，CRF刺激的腺苷酸环化酶活性被GTP增强，而在表达CRF-RII的细胞膜中，CRF刺激的腺苷酸环化酶活性对GTP不敏感。这些数据表明CRF-RII与G蛋白的偶联不佳。由于CRF-RII和CRF-RI之间的唯一区别是第一个假定的细胞质环中的插入片段，这些数据表明第一个细胞质环在hCRF受体与G蛋白的偶联中起关键作用。
