The Proto-oncogene SET Interacts with Muscarinic Receptors and Attenuates Receptor Signaling

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 ϳ35% 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. 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 biogenic amines, peptides, glycoproteins, lipids, nucleotides, ions, proteases, photons, odors, and taste. GPCRs possess a characteristic seven transmembrane domains linked by three extracellular and three intracellular loops. Interaction of agonist with the receptor initiates conformational changes within the receptor that are propagated to intracellular domains of the receptor, resulting in G protein activation and initiation of intracellular events. A long-term objective of our research effort is to define factors that influence the specificity and efficiency of signal propagation from GPCRs to G protein and perhaps function in the context of a larger signal transduction complex. Such proteins may influence receptor targeting to specific subcellular compartments, assembly of the receptor into functional complexes (i.e. receptosomes), and receptor trafficking to and from the plasma membrane as well as effector activation (1, 2).The third intracellular loop (i3 loop) and the carboxyl terminus tail of the receptor are key sites for signal initiation and termination for most members of GPCRs. These regions can be of considerable size and represent potential sites for protein interaction.Carboxyl-terminal domains of GPCRs contain discrete motifs that efficiently interact with cellular proteins such as the PDZ (PSD95-disc large-Zonula occludens) recognition motif (3). Other proteins interact with the carboxyl termini of some GPCRs via Src homology...

Muscarinic M4 Receptor Recycling Requires a Motif in the Third Intracellular Loop

“…On the other hand, the jpet.aspetjournals.org central part of i3 appears to be responsible for regulation of receptors. The i3 of M2 has been shown to be the site for phosphorylation (Nakata et al, 1994) and for interaction with G␤␥ (Wu et al, 1998); i3 of M3 has been shown to be the site for interaction with proteins, such as ␤-arrestin, G␤␥, casein kinase 1␣, and SET (Wu et al, 1997(Wu et al, , 2000Budd et al, 2000;Simon et al, 2006); i3 of M1 has been shown to be the site for interaction with regulators of G protein signaling (Bernstein et al, 2004); and i3 of M4 has been shown to be the site for interaction with elongation factor (McClatchy et al, 2002).…”

SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells

“…A similar inhibitory action of SET on M3-MR coupling to calcium signaling has been reported (18). Interestingly, a recent study addressing some of the mechanisms involved in SET action on M3-MR signaling showed that SET decreased G q protein engagement by the M3-MR, possibly through a competition for binding to the receptor, given the close proximity of the two protein binding sites within ICL3 (31) (40,41), suggesting that SET may also inhibit G q protein binding to GnRHR in gonadotrope ␣T3-1 cells.…”

“…SET, also called I2PP2A (inhibitor 2 of protein phosphatase 2A), is also an inhibitor of protein phosphatase 2A (PP2A) activity (22), a phosphatase involved in various signaling cascades (23). More recently, SET has been found to inhibit M3-MR coupling to the G q /PLC/ calcium pathway (18). Altogether, these data led us to hypothesize that SET may bind to GnRHR intracellular domains and influence its signaling.…”

“…There is now growing evidence that SET is an accessory protein for GPCR signaling. Its interaction was first reported with the ICL3 of the M3-MR (18). Since then, other receptors have been found to interact with SET (i.e.…”

“…Interestingly, in silico analysis of intracellular domain sequences of this receptor revealed the presence of basic amino acid clusters similar to the one previously identified within the muscarinic receptor. This basic amino acid cluster is located within the third intracellular loop of the M3 muscarinic receptor (M3-MR) and has been shown to interact with the protooncogene SET (18). SET was first described as part of the SET-CAN fusion gene, a putative oncogene associated with acute undifferentiated leukemia (19).…”