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.

Keywords: Cyclic AMP (cAMP); ERK; Endocytosis; G Protein-coupled Receptors (GPCR); Receptor Endocytosis; Signal Transduction.

FIGURE 1.

CRFR1-CT binds to a specific subset of PDZ proteins. Equal amounts of purified His-tagged fusion proteins corresponding to a subset of PDZ domains were spotted on nylon membranes and overlaid with GST (A) versus GST-CRFR1-CT (B) to reveal specific CRFR1-CT binding to the spotted PDZ domains. C, identity of 96 distinct PDZ domains that were spotted on the nylon membranes. Data are representative of four independent experiments.

FIGURE 2.

GFP-SAP97 co-immunoprecipitates with HA-CRFR1 in a PDZ-binding motif-dependent and CRF agonist-independent manner. A, representative immunoblot of SAP97 co-immunoprecipitated (IP) with HA-CRFR1 but not HA-CRFR1ΔTAV. Transient transfections were performed in HEK 293 cells as labeled. Samples were run using SDS-PAGE and immunoblotted (IB) with rabbit anti-GFP. GFP-SAP97 co-immunoprecipitated with HA-CRFR1 but not HA-CRFR1ΔTAV, which lacks the PDZ-binding motif. B, effect of CRF treatment was quantified using densitometry and had no significant effect on the amount of GFP-SAP97 co-immunoprecipitated with HA-CRFR1. Data are representative of six independent experiments. C, representative immunoblot for endogenous SAP97 co-immunoprecipitated with endogenous CRFR1 but not CRFR2 from 2 mg of mouse cortical lysate. SAP97, CRFR1, and CRFR2 expression in 100 μg of cortical lysate are shown below. Data are representative of three independent experiments.

FIGURE 3.

GFP-SAP97 co-localizes at the membrane with HA-CRFR1 in a PDZ-binding motif-dependent manner. A, representative confocal microscopy image demonstrating the co-localization of GFP-SAP97 (green) and cell surface HA-CRFR1 (red) labeled with Zenon Alexa Fluor 633-conjugated mouse HA antibody in live HEK 293 cells. B, representative confocal microscopy image demonstrating the co-localization of GFP-SAP97 (green) and cell surface HA-CRFR1-ΔTAV (red) labeled with Zenon Alexa Fluor 633-conjugated mouse HA antibody in live HEK 293 cells. C, quantification of CRFR and GFP-SAP97 co-localization. Data are representative of 37 (HA-CRFR1) and 17 (HA-CRFR1-ΔTAV) cells. *, p < 0.05.

FIGURE 4.

SAP97 antagonizes HA-CRFR1 endocytosis. A, agonist-stimulated internalization of either HA-CRFR1 or HA-CRFR1-ΔTAV in cells co-transfected with either GFP or GFP-SAP97. The internalization of HA-tagged receptors labeled with Alexa Fluor-conjugated mouse anti-NA antibody was measured in cells treated with 500 nm CRF for 30 min and compared with vehicle-treated control cells. The data represent the mean ± S.E. of nine independent experiments. *, p < 0.05 versus control CRFR1 internalization. B, representative immunoblot of endogenous SAP97 protein expression in HEK 293 cells transfected with 3 μg of plasmid cDNA encoding either scrambled (SCR) or SAP97 shRNA at 48 and 72 h initial transfection. IB, immunoblot. C, agonist-stimulated (500 nm CRF) internalization of HA-CRFR1 in cells co-transfected with scrambled (SCR) and SAP shRNA at 5, 15, 30, and 60 min. The data represent the mean ± S.E. of five independent experiments. *, p < 0.05 versus SCR shRNA-treated cells. D, agonist-stimulated (500 nm CRF) internalization of HA-CRFR1-ΔTAV in cells co-transfected with scrambled (SCR) and SAP97 shRNA at 30 and 60 min. The data represent the mean ± S.E. of four independent experiments.

FIGURE 5.

GFP-SAP97 exhibits limited endocytosis with HA-CRFR1. Live cell microscopic imaging of GFP-SAP97 (green) and HA-CRFR1 (red) labeled with Alexa Fluor 633-conjugated mouse anti-HA antibody prior (A) to and following (B) 500 nm activation for 30 min in a live HEK 293 cell is shown. Image is representative of 20 cells.

FIGURE 6.

SAP97 does not regulate CRFR1-mediated cAMP formation. A, CRFR1-mediated cAMP formation, as assessed by a BRET-based biosensor assay, following co-transfection with either GFP (control) or GFP-SAP97. The data represent the mean ± S.E. of seven independent experiments. B, CRFR1- and CRFR1-ΔTAV-mediated cAMP formation, as assessed by a BRET-based biosensor assay. The data represent the mean ± S.E. of four independent experiments. C, CRFR1-mediated cAMP formation as assessed by a cAMP GLO assay following co-transfection with either scrambled (SCR) or SAP97 shRNA. The data represent the mean ± S.E. of five independent experiments.

FIGURE 7.

Knockdown of endogenous SAP97 suppresses HA-CRFR1-mediated ERK1/2 phosphorylation. A, representative immunoblot showing ERK1/2 phosphorylation in response to 500 nm CRF treatment for 0, 2, 5, 15, and 30 min in nontransfected (NT) HEK 293 cells and HEK 293 cells transfected with HA-CRFR1 and either scrambled (SCR) or SAP97 shRNA. Shown are the corresponding immunoblots for total ERK1/2, SAP97, and HA-CRFR1 protein expression. B, densitometric analysis of ERK1/2 phosphorylation in response to 500 nm CRF treatment for 0, 2, 5, 15, and 30 min in nontransfected (NT) HEK 293 cells, and HEK 293 cells transfected with HA-CRFR1 and either scrambled (SCR) or SAP97 shRNA. The data represent the mean ± S.E. of four independent experiments. *, p < 0.05 versus SCR shRNA-treated cells. C, representative immunoblot (IB) showing ERK1/2 phosphorylation in response 500 nm CRF treatment for 0, 5, and 10 min in AtT20 cells transfected with either scrambled (SCR) or SAP97 siRNA. Shown are the corresponding immunoblots for total ERK1/2 and SAP97 protein expression. D, densitometric analysis of ERK1/2 phosphorylation in response to 500 nm CRF treatment for 0, 5, and 10 min in AtT20 cells transfected with either scrambled (SCR) or SAP97 siRNA. The data represent the mean ± S.E. of four independent experiments. *, p < 0.05 versus SCR shRNA-treated cells.

FIGURE 8.

Knockdown of endogenous SAP97 suppresses HA-CRFR1-ΔTAV- and HA-CRFR2-mediated ERK1/2 phosphorylation. A, representative immunoblot (IB) showing ERK1/2 phosphorylation in response to 500 nm CRF treatment for 0, 2, and 5 min in HEK 293 cells transfected with HA-CRFR1-ΔTAV and either scrambled (SCR) or SAP97 shRNA. Shown are the corresponding immunoblots for total ERK1/2, SAP97, and HA-CRFR1-ΔTAV protein expression. B, densitometric analysis of ERK1/2 phosphorylation in response to 500 nm CRF treatment for 0, 2, and 5 min in HEK 293 cells transfected with HA-CRFR1-ΔTAV and either scrambled (SCR) or SAP97 shRNA. The data represent the mean ± S.E. of four independent experiments. *, p < 0.05 versus SCR shRNA-treated cells. C, representative immunoblot showing ERK1/2 phosphorylation in response to 500 nm CRF treatment for 0, 2, 5, 15, and 30 min in nontransfected (NT) HEK 293 cells and HEK 293 cells transfected with HA-CRFR2 and either scrambled (SCR) or SAP97 shRNA. Shown are the corresponding immunoblots for total ERK1/2, SAP97, and HA-CRFR2 protein expression. D, densitometric analysis of ERK1/2 phosphorylation in response to 500 nm CRF treatment for 0, 2, 5, 15, and 30 min in nontransfected (NT) HEK 293 cells and HEK 293 cells transfected with HA-CRFR2 and either scrambled (SCR) or SAP97 shRNA. The data represent the mean ± S.E. of three independent experiments. *, p < 0.05 versus SCR shRNA-treated cells.