title: Role of SAP97 in the regulation of 5-HT2AR endocytosis and signaling.
abstract: Serotonin (5-HT) interacts with a wide variety of 5-HT receptors (5-HTR) of which 5-HT2AR plays an important target for antidepressant and atypical antipsychotic drugs. The carboxyl-terminal tail of 5-HT2AR encodes a motif that mediates interactions with PSD-95/disc large/zona occludens (PDZ) domain-containing proteins. In the present study, we found that 5-HT2AR interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by coimmunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates. We found that 5-HT2AR expression results in the recruitment of SAP97 from the cytosol to the plasma membrane and that this recruitment is dependent on an intact 5-HT2AR PDZ binding motif. We also show that 5-HT2AR interacts with SAP97 using bioluminescence energy transfer and that overexpression of SAP97 retards 5-HT2AR endocytosis, while single hairpin RNA knockdown facilitates 5-HT2AR internalization. The knockdown of SAP97 in HEK 293 cells results in a reduction in the maximum efficacy for 5-HT2AR-stimulated inositol phosphate formation and that the deletion of the 5-HT2AR PDZ motif also impairs 5-HT2AR signaling. Similarly to what has been observed for the corticotropin-releasing factor receptor 1 (CRFR1), SAP97 expression is essential for 5-HT2AR-stimulated extracellular-regulated protein kinase 1/2 (ERK1/2) phosphorylation by a PDZ interaction-independent mechanism. Moreover, we find that SAP97 is not responsible for CRFR1-mediated sensitization of 5-HT2AR signaling. Taken together, our studies show that SAP97 plays a conserved role in regulating 5-HT2AR endocytosis and ERK1/2 signaling, but plays a novel role in regulating 5-HT2AR G protein coupling.
authors.affiliation: J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute and the Department of Physiology and Pharmacology, University of Western Ontario, Ontario, Canada.
authors.forename: Henry A
authors.initials: HA
authors.lastname: Dunn
authors.affiliation: J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute and the Department of Physiology and Pharmacology, University of Western Ontario, Ontario, Canada.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Walther
authors.affiliation: J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute and the Department of Physiology and Pharmacology, University of Western Ontario, Ontario, Canada.
authors.forename: George Y
authors.initials: GY
authors.lastname: Yuan
authors.affiliation: J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute and the Department of Physiology and Pharmacology, University of Western Ontario, Ontario, Canada.
authors.forename: Fabiana A
authors.initials: FA
authors.lastname: Caetano
authors.affiliation: J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute and the Department of Physiology and Pharmacology, University of Western Ontario, Ontario, Canada.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Godin
authors.affiliation: J. Allyn Taylor Centre for Cell Biology, Robarts Research Institute and the Department of Physiology and Pharmacology, University of Western Ontario, Ontario, Canada ferguson@robarts.ca.
authors.forename: Stephen S G
authors.initials: SS
authors.lastname: Ferguson
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D000074288:Discs Large Homolog 1 Protein; D004705:Endocytosis; D004789:Enzyme Activation; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D008565:Membrane Proteins / Q000502:physiology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D044402:Receptor, Serotonin, 5-HT2A / Q000502:physiology*; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism; D015398:Signal Transduction; D000099052:CRF Receptor, Type 1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C513314:DLG1 protein, human; D000074288:Discs Large Homolog 1 Protein; D007295:Inositol Phosphates; D008565:Membrane Proteins; D044402:Receptor, Serotonin, 5-HT2A; D018019:Receptors, Corticotropin-Releasing Hormone; D000099052:CRF Receptor, Type 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3