title: 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.
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-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.
authors.affiliation: Endocrinology and Metabolism, Clinical Sciences Institute, Warwick Medical School, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, United Kingdom.
authors.forename: Danijela
authors.initials: D
authors.lastname: Markovic
authors.forename: Hendrik
authors.initials: H
authors.lastname: Lehnert
authors.forename: Michael A
authors.initials: MA
authors.lastname: Levine
authors.forename: Dimitris K
authors.initials: DK
authors.lastname: Grammatopoulos
mesh_terms: D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003346:Corticotropin-Releasing Hormone / Q000378:metabolism / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004705:Endocytosis; D005091:Exons; D005260:Female; D014644:Genetic Variation; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009215:Myometrium / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018019:Receptors, Corticotropin-Releasing Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D054832:Urocortins / Q000378:metabolism / Q000494:pharmacology; D000099052:CRF Receptor, Type 1
chemical_list: C101531:CRF receptor type 2; D017931:DNA Primers; D012333:RNA, Messenger; D018019:Receptors, Corticotropin-Releasing Hormone; D011994:Recombinant Proteins; C426705:UCN2 protein, human; D054832:Urocortins; D000099052:CRF Receptor, Type 1; D003346:Corticotropin-Releasing Hormone