title: Role of SAP97 protein in the regulation of corticotropin-releasing factor receptor 1 endocytosis and extracellular signal-regulated kinase 1/2 signaling.
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.
authors.affiliation: J Allyn Taylor Centre for Cell Biology, Robarts Research Institute, University of Western Ontario, London, Ontario N6A 5K8, Canada.
authors.forename: Henry A
authors.initials: HA
authors.lastname: Dunn
authors.forename: Cornelia
authors.initials: C
authors.lastname: Walther
authors.forename: Christina M
authors.initials: CM
authors.lastname: Godin
authors.forename: Randy A
authors.initials: RA
authors.lastname: Hall
authors.forename: Stephen S G
authors.initials: SS
authors.lastname: Ferguson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D000074288:Discs Large Homolog 1 Protein; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000235:genetics / Q000378:metabolism; D051528:Guanylate Kinases / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D018019:Receptors, Corticotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; D000074288:Discs Large Homolog 1 Protein; C000614189:Dlg1 protein, mouse; C501119:Dlg1 protein, rat; D008565:Membrane Proteins; D018019:Receptors, Corticotropin-Releasing Hormone; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D051528:Guanylate Kinases
keywords: Cyclic AMP (cAMP); ERK; Endocytosis; G Protein-coupled Receptors (GPCR); Receptor Endocytosis; Signal Transduction