Role of SAP97 protein in the regulation of corticotropin-releasing factor receptor 1 endocytosis and extracellular signal-regulated kinase 1/2 signaling.
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.
['Dunn|Henry A|HA|', 'Walther|Cornelia|C|', 'Godin|Christina M|CM|', 'Hall|Randy A|RA|', 'Ferguson|Stephen S G|SS|']
D048868:Adaptor Proteins, Signal Transducing D020816:Amino Acid Motifs D000818:Animals D002462:Cell Membrane D000074288:Discs Large Homolog 1 Protein D004705:Endocytosis D011992:Endosomes D051528:Guanylate Kinases D057809:HEK293 Cells D006801:Humans D020935:MAP Kinase Signaling System D008565:Membrane Proteins D019950:Mitogen-Activated Protein Kinase 1 D048052:Mitogen-Activated Protein Kinase 3 D010766:Phosphorylation D021381:Protein Transport D051381:Rats D018019:Receptors, Corticotropin-Releasing Hormone
Endocytosis Cyclic AMP (cAMP) ERK G Protein-coupled Receptors (GPCR) Receptor Endocytosis Signal Transduction