The proto-oncogene SET interacts with muscarinic receptors and attenuates receptor signaling.
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.
Simon
Violaine
V
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Guidry
Jessie
J
Gettys
Thomas W
TW
Tobin
Andrew B
AB
Lanier
Stephen M
SM
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002868:Chromosomal Proteins, Non-Histone / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D006224:Cricetinae; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D043585:Receptor, Muscarinic M2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D043587:Receptor, Muscarinic M3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D014157:Transcription Factors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D056488:Histone Chaperones; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D043562:Receptors, G-Protein-Coupled; C513473:SET protein, human; D014157:Transcription Factors