Markovic Danijela, Lehnert Hendrik, Levine Michael A, Grammatopoulos Dimitris K
Endocrinology and Metabolism, Clinical Sciences Institute, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom.
Mol Endocrinol. 2008 Nov;22(11):2505-19. doi: 10.1210/me.2008-0177. Epub 2008 Sep 4.
The type 1 CRH receptor (CRH-R1) plays a fundamental role in homeostatic adaptation to stressful stimuli. CRH-R1 gene activity is regulated through alternative splicing and generation of various CRH-R1 mRNA variants. One such variant is the CRH-R1d, which has 14 amino acids missing from the putative seventh transmembrane domain due to exon 13 deletion, a splicing event common to other members of the B1 family of G protein-coupled receptors. In this study, using overexpression of recombinant receptors in human embryonic kidney 293 and myometrial cells, we showed by confocal microscopy that in contrast to CRH-R1alpha, the R1d variant is primarily retained in the cytoplasm, although some cell membrane expression is also evident. Use of antibodies against the CRH-R1 C terminus in nonpermeabilized cells showed that membrane-expressed CRH-R1d contains an extracellular C terminus. Interestingly, treatment of CRH-R1d-expressing cells with CRH (100 nM) for 45-60 min elicited functional responses associated with a significant reduction of plasma membrane receptor expression, redistribution of intracellular receptors, and increased receptor degradation. Site-directed mutagenesis studies identified the cassette G356-F358 within transmembrane domain 7 as crucial for CRH-R1alpha stability to the plasma membrane because deletion of this cassette caused substantial intracellular localization of CRH-R1 alpha. Most importantly, coexpression studies between CRH-R1d and CRH-R2beta demonstrated that the CRH-R2beta could partially rescue CRH-R1d membrane expression, and this was associated with a significant attenuation of urocotrin II-induced cAMP production and ERK1/2 and p38MAPK activation, suggesting that CRH-R1d might specifically induce heterologous impairment of CRH-R2 signaling responses. This mechanism appears to involve accelerated CRH-R2beta endocytosis.
1型促肾上腺皮质激素释放激素受体（CRH-R1）在对应激刺激的稳态适应中起重要作用。CRH-R1基因活性通过可变剪接和产生多种CRH-R1 mRNA变体来调节。其中一种变体是CRH-R1d，由于外显子13缺失，其假定的第七跨膜结构域缺少14个氨基酸，这是G蛋白偶联受体B1家族其他成员常见的剪接事件。在本研究中，我们通过在人胚肾293细胞和子宫肌层细胞中过表达重组受体，利用共聚焦显微镜显示，与CRH-R1α相反，R1d变体主要保留在细胞质中，尽管也有一些细胞膜表达。在未通透细胞中使用针对CRH-R1 C末端的抗体表明，膜表达的CRH-R1d含有细胞外C末端。有趣的是，用促肾上腺皮质激素释放激素（CRH，100 nM）处理表达CRH-R1d的细胞45 - 60分钟会引发功能反应，这与质膜受体表达显著减少以及细胞内受体重新分布和受体降解增加有关。定点诱变研究确定跨膜结构域7内的G356 - F358盒对CRH-Rα定位于质膜的稳定性至关重要，因为缺失该盒会导致CRH-R1α大量定位于细胞内。最重要的是，CRH-R1d和CRH-R2β之间的共表达研究表明，CRH-R2β可以部分挽救CRH-R1d的膜表达，这与尿皮质素II诱导的cAMP产生以及ERK1/2和p38MAPK激活的显著减弱有关，表明CRH-R1d可能特异性诱导CRH-R2信号反应的异源损伤。这种机制似乎涉及加速CRH-R2β的内吞作用。
