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Structural Determinants Critical for Localization and Signaling within the Seventh Transmembrane Domain of the Type 1 Corticotropin Releasing Hormone Receptor: Lessons from the Receptor Variant R1d

Endocrinology and Metabolism (D.M., H.L., D.K.G.), Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV4 7AL, United Kingdom; and Division of Pediatrics and Diabetes (M.A.L.), The Children’s Hospital of Philadelphia and the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

Address all correspondence and requests for reprints to: Professor D.K Grammatopoulos, Sir Quinton Hazell Molecular Medicine Research Centre, Warwick Medical School, The University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom. E-mail: d.grammatopoulos{at}warwick.ac.uk.

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-R1, 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-R1 stability to the plasma membrane because deletion of this cassette caused substantial intracellular localization of CRH-R1 . Most importantly, coexpression studies between CRH-R1d and CRH-R2β demonstrated that the CRH-R2β 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-R2β endocytosis.

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