Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling

• Type of Material: Article
• Authors: Randy A Hall, Emory University; Henry A. Dunn, University of Western Ontario; Cornelia Walther, University of Western Ontario; Christina M. Godin, University of Western Ontario; Stephen S. G. Ferguson, University of Western Ontario
• Language: English
• Date: 2013-05-24
• Publisher: American Society for Biochemistry and Molecular Biology
• Publication Version: Final Published Version
• Copyright Statement: © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.
• Final Published Version (URL): http://dx.doi.org/10.1074/jbc.M113.473660
• Title of Journal or Parent Work: Journal of Biological Chemistry
• ISSN: 0021-9258
• Volume: 288
• Issue: 21
• Start Page: 15023
• End Page: 15034
• Grant/Funding Information: This work was supported by Canadian Institutes of Health Research Grant MOP-62738 (to S. S. G. F.).
• Keywords: DESENSITIZATION; INTERACTING PROTEINS; PHOSPHORYLATION; BETA(1)-ADRENERGIC RECEPTOR; Science & Technology; Life Sciences & Biomedicine; PEPTIDE FAMILY; SCAFFOLD; Biochemistry & Molecular Biology; PDZ PROTEINS; TYPE-1 RECEPTOR; RAT-BRAIN; DIFFERENTIAL REGULATION
• Research Categories: Biology, Cell; Health Sciences, Pharmacology

Abstract

The corticotropin-releasing factor (CRF) receptor 1 (CRFR1) is a target for the treatment of psychiatric diseases such as depression, schizophrenia, anxiety disorder, and bipolar disorder. The carboxyl-terminal tail of the CRFR1 terminates in a PDZ-binding motif that provides a potential site for the interaction of PSD-95/Discs Large/Zona Occludens 1 (PDZ) domain-containing proteins. In this study, we found that CRFR1 interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by co-immunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates and that this interaction is dependent upon an intact PDZ-binding motif at the end of the CRFR1 carboxyl-terminal tail. Similarly, we demonstrated that SAP97 is recruited to the plasma membrane in HEK 293 cells expressing CRFR1 and that mutation of the CRFR1 PDZ-binding motif results in the redistribution of SAP97 into the cytoplasm. Overexpression of SAP97 antagonized agonist-stimulated CRFR1 internalization, whereas single hairpin (shRNA) knockdown of endogenous SAP97 in HEK 293 cells resulted in increased agonist-stimulated CRFR1 endocytosis. CRFR1 was internalized as a complex with SAP97 resulting in the redistribution of SAP97 to endocytic vesicles. Overexpression or shRNA knockdown of SAP97 did not significantly affect CRFR1-mediated cAMP formation, but SAP97 knockdown did significantly attenuate CRFR1-stimulated ERK1/2 phosphorylation in a PDZ interaction-independent manner. Taken together, our studies show that SAP97 interactions with CRFR1 attenuate CRFR1 endocytosis and that SAP97 is involved in coupling G protein-coupled receptors to the activation of the ERK1/2 signaling pathway.