Abstract
G protein-coupled receptors mediate cell responses to extracellular stimuli and likely function in the context of a larger signal transduction complex. Utilizing the third intracellular loop of a G protein-coupled receptor in glutathione S-transferase pulldown assays from rat brain lysates coupled with high sensitivity detection methods and subsequent functional studies, we report the identification of SET as a regulator of muscarinic receptor signaling. SET is a putative oncogene reported to inhibit protein phosphatase 2A and regulate gene transcription. SET binds the carboxyl region of the M3-muscarinic receptor i3 loop, and endogenous SET co-immunoprecipitates with intact M3 muscarinic receptor expressed in cells. Small interfering RNA knockdown of endogenous SET in Chinese hamster ovary cells stably expressing the M3 muscarinic receptor augmented receptor-mediated mobilization of intracellular calcium by approximately 35% with no change in agonist EC(50), indicating that interaction of SET with the M3 muscarinic receptor reduces its signaling capacity. SET knockdown had no effect on the mobilization of intracellular calcium by the P2-purinergic receptor, ionomycin, or a direct activator of phospholipase C, indicating a specific regulation of M3 muscarinic receptor signaling. These data provide expanded functionality for SET and a previously unrecognized mechanism for regulation of GPCR signaling capacity.
Highlights
G protein-coupled receptors (GPCRs)2 represent the largest family of membranous receptors and process signals from a great diversity of endogenous and exogenous stimuli including
Utilizing the third intracellular loop of a G protein-coupled receptor in glutathione S-transferase pulldown assays from rat brain lysates coupled with high sensitivity detection methods and subsequent functional studies, we report the identification of SET as a regulator of muscarinic receptor signaling
Other proteins interact with the carboxyl termini of some GPCRs via Src homology 2 (SH2) and SH3, pleckstrin homology, or Ena/VASP homology (EVH) domains [3]
Summary
Recombinant Protein Preparations—The cDNAs encoding the i3 loop of the human M2- and rat M3-MRs (His208–Arg387 and Arg252–Gln490, respectively) were subcloned into pGEX-2T or pGEX-4T-1 vectors, respectively [4]. The resin was pelleted, washed 3 times with Nonidet P-40 lysis buffer, resuspended in 2ϫ protein sample buffer, and incubated at room temperature for 30 min followed by SDS-PAGE and immunoblotting with polyclonal anti-M3MR antibody (provided by Dr Jurgen Wess, National Institutes of Health, Bethesda) [16] and SET antibody. To assess SET siRNA knockdown, cells were homogenized in lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40) and incubated for 1 h on ice. Cell homogenates were centrifuged at 10,000 ϫ g at 4 °C, and 12.5 g of the supernatant were separated on a 10% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride membrane. 4 –7 independent experiments; the data point for any drug concentration is an average from 3 to 4 wells
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
