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.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Amino Acid Motifs, Animals, Cell Membrane, Discs Large Homolog 1 Protein, Endocytosis, Endosomes, Guanylate Kinases, HEK293 Cells, Humans, MAP Kinase Signaling System, Membrane Proteins, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Phosphorylation, Protein Transport, Rats, Receptors, Corticotropin-Releasing Hormone
Adaptor Proteins, Signal Transducing, Amino Acid Motifs, Animals, Cell Membrane, Discs Large Homolog 1 Protein, Endocytosis, Endosomes, Guanylate Kinases, HEK293 Cells, Humans, MAP Kinase Signaling System, Membrane Proteins, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Phosphorylation, Protein Transport, Rats, Receptors, Corticotropin-Releasing Hormone
J. Biol. Chem.
Date: May. 24, 2013
PubMed ID: 23576434
View in: Pubmed Google Scholar
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