Markovic, Danijela, Lehnert, Hendrik, Levine, Michael A. and Grammatopoulos, Dimitris K.. (2008) 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. Molecular Endocrinology, Vol.22 (No.11). pp. 2505-2519. ISSN 0888-8809

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Abstract

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 alpha, 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 G(356)- F-358 within transmembrane domain 7 as crucial for CRH-R1 alpha 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-R2 beta demonstrated that the CRH-R2 beta 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-R2 beta endocytosis. (Molecular Endocrinology 22: 2505-2519, 2008)

Item Type:	Journal Article
Subjects:	R Medicine > RC Internal medicine
Divisions:	Faculty of Medicine > Warwick Medical School > Metabolic and Vascular Health Faculty of Medicine > Warwick Medical School
Journal or Publication Title:	Molecular Endocrinology
Publisher:	Endocrine Society
ISSN:	0888-8809
Date:	November 2008
Volume:	Vol.22
Number:	No.11
Number of Pages:	15
Page Range:	pp. 2505-2519
Identification Number:	10.1210/me.2008-0177
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Funder:	Wellcome Trust University Award
URI:	http://wrap.warwick.ac.uk/id/eprint/29120

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