Abstract
The 5-hydroxytryptamine 2A (5-HT(2A)) receptor is a member of the G protein-coupled receptor superfamily (GPCR) and plays a key role in transducing a variety of cellular signals elicited by 5-hydroxytryptamine in both peripheral and central tissues. Despite its broad physiological importance, our current understanding of 5-HT(2A) receptor regulation is incomplete. We recently reported the novel finding that the multifunctional ERK effector ribosomal S6 kinase 2 (RSK2) physically interacts with the 5-HT(2A) receptor third intracellular (i3) loop and modulates receptor signaling (Sheffler, D. J., Kroeze, W. K., Garcia, B. G., Deutch, A. Y., Hufeisen, S. J., Leahy, P., Bruning, J. C., and Roth, B. L. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 4717-4722). We report here that RSK2 directly phosphorylates the 5-HT(2A) receptor i3 loop at the conserved residue Ser-314, thereby modulating 5-HT(2A) receptor signaling. Furthermore, these studies led to the discovery that RSK2 is required for epidermal growth factor-mediated heterologous desensitization of the 5-HT(2A) receptor. We arrived at these conclusions via multiple lines of evidence, including in vitro kinase experiments, tandem mass spectrometry, and site-directed mutagenesis. Our findings were further validated using phospho-specific Western blot analysis, metabolic labeling studies, and whole-cell signaling experiments. These results support a novel regulatory mechanism in which a downstream effector of the ERK/MAPK pathway directly interacts with, phosphorylates, and modulates signaling of the 5-HT(2A) serotonin receptor. To our knowledge, these findings are the first to demonstrate that a downstream member of the ERK/MAPK cascade phosphorylates a GPCR as well as mediates cross-talk between a growth factor and a GPCR.
Highlights
The 5-HT2A2 receptor plays a key role in transducing a variety of cellular signals elicited by 5-HT in both peripheral and
Extensive studies focusing on the G protein-coupled receptor kinase-arrestin pathway and the second messenger-dependent protein kinase (cAMPdependent protein kinase and protein kinase C (PKC)) pathways suggest that direct G protein-coupled receptor superfamily (GPCR) phosphorylation remains the predominant mechanism for rapidly attenuating the signaling of many GPCRs (9, 10)
ribosomal S6 kinase 2 (RSK2) N-terminal Kinase Activity Is Required for 5-hydroxytryptamine 2A (5-HT2A) Receptor Regulation—We have previously shown that the Ser/ Thr kinase RSK2 interacts with the 5-HT2A receptor i3 loop in a conserved region containing the RSK2-like phosphorylation motif 275RAKL(A/S)S280 (21)
Summary
Materials—Cell culture reagents were supplied by Invitrogen and Cambrex (East Rutherford, NJ). Site-directed Mutagenesis—The QuikChange site-directed mutagenesis kit (Stratagene) was used to generate kinase-dead RSK2 mutants, i3 loop peptide mutants, and a full-length FLAG-His6 5-HT2A receptor mutant using the wild-type constructs as PCR templates. In Vitro Kinase Assays—In vitro kinase assays included the following: 1) purified, activated RSK2 (Upstate-Millipore, Billerica, MA) diluted in enzyme buffer (20 mM MOPS, 1 mM EGTA, 0.01% Brij-35, 5% glycerol, 0.1% 2-mercaptoethanol, 1 mg/ml BSA, pH 7.5); 2) i3 loop peptide or FLAG-His6 5-HT2A receptor substrates; and 3) [␥-32P]ATP or unlabeled ATP diluted in assay buffer (75 mM MgCl2, 20 mM MOPS, 25 mM -glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, pH 7.2). We assayed for [32P]phosphate incorporation by incubating purified i3 loop peptide or purified FLAG-His6 5-HT2A receptor with 0.4 – 0.8 ng/l RSK2 in the presence of 0.4 – 0.04 Ci/l [␥-32P]ATP for 1 h at 30 °C. Statistical significance of IP3 time course data were determined by one-tailed paired t test and defined as p Ͻ 0.05
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
