Differentially Regulated Expression of Endogenous RGS4 and RGS7

Abstract

Regulators of G protein signaling (RGS proteins) constitute a family of newly appreciated components of G protein-mediated signal transduction. With few exceptions, most information available on mammalian RGS proteins was gained by transfection/overexpression or in vitro experiments, with relatively little known about the endogenous counterparts. Transfection studies, typically of tagged RGS proteins, have been conducted to overcome the low natural abundance of endogenous RGS proteins. Because transfection studies can lead to imprecise or erroneous conclusions, we have developed antibodies of high specificity and sensitivity to focus study on endogenous proteins. Expression of both RGS4 and RGS7 was detected in rat brain tissue and cultured PC12 and AtT-20 cells. Endogenous RGS4 presented as a single 27–28-kDa protein. By contrast, cultured cells transfected with a plasmid encoding RGS4 expressed two observable forms of the protein, apparently due to utilization of distinct sites of initiation of protein synthesis. Subcellular localization of endogenous RGS4 revealed predominant association with membrane fractions, rather than in cytosolic fractions, where most heterologously expressed RGS4 has been found. Endogenous levels of RGS7 exceeded RGS4 by 30–40-fold, and studies of cultured cells revealed regulatory differences between the two proteins. We observed that RGS4 mRNA and protein were concomitantly augmented with increased cell density and decreased by exposure of PC12M cells to nerve growth factor, whereas RGS7 was unaffected. Endogenous RGS7 was relatively stable, whereas proteolysis of endogenous RGS4 was a strong determinant of its lower level expression and short half-life. Although we searched without finding evidence for regulation of RGS4 proteolysis, the possibility remains that alterations in the degradation of this protein could provide a means to promptly alter patterns of signal transduction. Regulators of G protein signaling (RGS proteins) constitute a family of newly appreciated components of G protein-mediated signal transduction. With few exceptions, most information available on mammalian RGS proteins was gained by transfection/overexpression or in vitro experiments, with relatively little known about the endogenous counterparts. Transfection studies, typically of tagged RGS proteins, have been conducted to overcome the low natural abundance of endogenous RGS proteins. Because transfection studies can lead to imprecise or erroneous conclusions, we have developed antibodies of high specificity and sensitivity to focus study on endogenous proteins. Expression of both RGS4 and RGS7 was detected in rat brain tissue and cultured PC12 and AtT-20 cells. Endogenous RGS4 presented as a single 27–28-kDa protein. By contrast, cultured cells transfected with a plasmid encoding RGS4 expressed two observable forms of the protein, apparently due to utilization of distinct sites of initiation of protein synthesis. Subcellular localization of endogenous RGS4 revealed predominant association with membrane fractions, rather than in cytosolic fractions, where most heterologously expressed RGS4 has been found. Endogenous levels of RGS7 exceeded RGS4 by 30–40-fold, and studies of cultured cells revealed regulatory differences between the two proteins. We observed that RGS4 mRNA and protein were concomitantly augmented with increased cell density and decreased by exposure of PC12M cells to nerve growth factor, whereas RGS7 was unaffected. Endogenous RGS7 was relatively stable, whereas proteolysis of endogenous RGS4 was a strong determinant of its lower level expression and short half-life. Although we searched without finding evidence for regulation of RGS4 proteolysis, the possibility remains that alterations in the degradation of this protein could provide a means to promptly alter patterns of signal transduction. G proteins transduce signals across the plasma membrane by sequential interactions with cell surface receptors and appropriate second messenger-producing effectors (e.g. enzymes and ion channels). These interactions are modulated by nucleotide-driven conformational changes in the α subunits of heterotrimeric G proteins (Gα). 1The abbreviations used are: Gα, α subunit of heterotrimeric G protein; RGS, regulator of G protein signaling; Gβγ, βγ subunits of heterotrimeric G protein; GAIP, Gα-interacting protein (also known as RGS19); GAP, GTPase-activating protein; NGF, nerve growth factor; siRNA, small interfering RNA. A ligand-bound receptor catalyzes the exchange of GDP for GTP on its cognate Gα and the dissociation of Gα from the complex of G protein β and γ subunits (Gβγ). These dissociated subunits are competent to modulate the activity of effectors. The duration of G protein-mediated responses are dependent on the intrinsic GTPase rate of Gα and on extrinsic factors, such as regulators of G protein signaling (RGS proteins). RGS proteins serve to regulate G protein signaling by functioning as GTPase-activating proteins (GAPs). GAP activity can sharpen the termination of a signal upon removal of a stimulus, attenuate a signal either as a feedback inhibitor or in response to a second input, promote regulatory association of other proteins, or redirect signaling within a G protein signaling network (reviewed in Ref. 1.Ross E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (929) Google Scholar). RGS proteins are related by a conserved RGS domain that is composed of ∼130 amino acid residues. The RGS domain alone is capable of binding Gα and accelerating GTP hydrolysis, although other domains contribute to affinity and/or selectivity for G protein targets (2.Patikoglou G.A. Koelle M.R. J. Biol. Chem. 2002; 277: 47004-47013Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 3.Zeng W. Xu Popov S. Mukhopadhyay S. Chidiac P. Swistok J. Danho W. Yagaloff K.A. Fisher S.L. Ross E.M. Muallem S. Wilkie T.M. J. Biol. Chem. 1998; 273: 34687-34690Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Mammalian RGS proteins, of which >20 are now known, can be grouped into five subfamilies based on sequence similarity (R4, R7, R12, RA, and RZ) (4.Sierra D.A. Popov S. Wilkie T.M. Trends Cardiovasc. Med. 2000; 10: 263-268Crossref PubMed Scopus (42) Google Scholar). Although several members of the RGS family are relatively simple ∼25-kDa proteins that contain short amino and carboxyl sequences flanking the characteristic RGS domain (such as RGS4), others include more substantial modules that impart other functions. The R7 subfamily is characterized by possession of so-called DEP (disheveled, EGL-10, pleckstrin) and GGL (G protein gamma subunit-like) domains. While not well established, the DEP domain may play a role in directing Gα subunit specificity for the RGS domain (2.Patikoglou G.A. Koelle M.R. J. Biol. Chem. 2002; 277: 47004-47013Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The GGL domain apparently specifies an obligate interaction of an RGS protein with the G protein β5 subunit (5.Snow B.E. Krumins A.M. Brothers G.M. Lee S.F. Wall M.A. Chung S. Mangion J. Arya S. Gilman A.G. Siderovski D.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13307-13312Crossref PubMed Scopus (229) Google Scholar, 6.Witherow D.S. Wang Q. Levay K. Cabrera J.L. Chen J. Willars G.B. Slepak V.Z. J. Biol. Chem. 2000; 275: 24872-24880Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). RGS4 has the capacity to accelerate in vitro GTPase activity of Gαi subfamily (including Gαi, Gαo, and Gαz) and Gαq subfamily members but not Gαs or Gα12 subfamily members. By contrast, GGL-containing RGS proteins exhibit specificity for Gαo and Gαt (7.Posner B.A. Gilman A.G. Harris B.A. J. Biol. Chem. 1999; 274: 31087-31093Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 8.He W. Cowan C.W. Wensel T.G. Neuron. 1998; 20: 95-102Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar). Much of the currently available information on mammalian RGS proteins was gained by transfection/overexpression or in vitro experiments, with little known about the endogenous counterparts (particularly for the RGS4 subfamily). Because such studies can lead to imprecise or erroneous conclusions, caused by problems such as mislocalization and/or loss of substrate specificity, we have focused study on endogenous proteins. A case in point is the apparent difference in selectivity of RGS2 (an R4 family member) for Gαi and Gαq when different transfection systems have been utilized (9.Ingi T. Krumins A. Chidiac P. Brothers G.M. Chung S. Snow B.E. Barnes C.A. Lanahan A.A. Siderovski D.P. Ross E.M. Gilman A.G. Worley P.F. J. Neurosci. 1998; 18: 7178-7188Crossref PubMed Google Scholar, 10.Heximer S.P. Srinivasa S.P. Bernstein L.S. Bernard J.L. Linder M.E. Hepler J.R. Blumer K.J. J. Biol. Chem. 1999; 274: 34253-34259Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Although the mRNA for RGS4 is relatively abundant in brain, detection of the protein only recently has been reported for this tissue (11.Muma N.A. Mariyappa R. Williams K. Lee J.M. Synapse. 2003; 47: PubMed Scopus Google Scholar). and degradation may the RGS4 protein low levels A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google the expression of substantial levels of and of endogenous RGS proteins, we have developed antibodies with appropriate specificity and sensitivity to endogenous RGS4 or we and two proteins with and differences between endogenous and heterologously of are in nerve growth and for expression of RGS4 short was by Wilkie T.M. Gilman A.G. Full Text Full Text PDF PubMed Scopus Google as a The an The was to a of the RGS4 termination within the expression plasmid The was with and and into a and The newly as the for RGS4 short into the mammalian expression S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and RGS4 was by of with or The were to of and expression was by for were in and as Wilkie T.M. Gilman A.G. Full Text Full Text PDF PubMed Scopus Google Scholar). RGS4 protein or expressed to levels than RGS4 and the predominant protein in the from high of were by and by A of RGS4 short and as a RGS4 was from the by and as for RGS4 Wilkie T.M. Gilman A.G. Full Text Full Text PDF PubMed Scopus Google Scholar). RGS4 from was into a for of an of protein was sites on the for the and of the a of was for to RGS7 were from with the the of to amino of The in was for to the protein, Gilman A.G. PubMed Scopus Google Scholar). antibodies were from the by binding to the on Gilman A.G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A was to a the amino of to the carboxyl of Gαi and has been M.E. P. Hepler J.R. R. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. Scopus Google Scholar). Mammalian and cells were from the of and of the of were cultured in with high with and an of A transfected of cells was as Hepler J.R. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of RGS4 and RGS7 in PC12M cells was by transfection of cells with and plasmid and/or short interfering to the The sequences of the of the used for RGS4 and RGS7 were and The to and of the were and the for the of transfected with were were with and were to and the were for cells were as Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). of of the rat brain were from were and the from the were for in were with an brain of or were to a on and were that were from the The tissue were by in and inhibitor and were for were for protein and the of the was on and and with protein was by W. Biochem. PubMed Scopus Google or by the J. Biol. Chem. Full Text PDF PubMed Google Scholar). not contain and were Biochem. PubMed Scopus Google Scholar). as in where of protein were by and was from PC12 cells The was and and into RGS4 was detected by of of and of for was on a and were detected with RGS4 as a single of with were by in a J. A Scholar). and of in well was by to and RNA. The was to membrane and with a the RGS The are is A of the is the of the RGS domain is within These on D.A. K. P. W. Wensel T.G. N.A. Wilkie T.M. 2002; PubMed Scopus Google and on were conducted the as by the a for an binding was by the where the binding is and a to of the RGS4 The used to the for the was as a plasmid for RGS4 and were and of in the The activity of the was for the A of the was to with of The was to A and and to of and for RGS4 and were on a were to were developed by a and were GAP activity in from PC12 cells was as as substrate J. J. Ross E.M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of cells transfected with a for RGS4 expressed a protein that more on than RGS4 protein from and the expression of an apparently than of RGS4 was not cell was observed in transfected cells and cells not We and others A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google that the sequence the encoding the second a PubMed Scopus Google and could the of the short of RGS4 in transfected mammalian cells. this RGS4 the encoding the amino were for expression in mammalian cells and The RGS4 short from with the RGS4 protein expressed in cells transfected with the or short RGS4 or RGS4 in the expression of a of the protein in cells and These that the heterologously which we can is the the Subcellular of RGS4 heterologously in cells was in the was the case RGS4 was tagged or or this to the localization of G protein signaling the plasma is with other Blumer K.J. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, A.A. Chidiac P. 2003; PubMed Scopus Google Scholar). endogenous RGS4 a we and was RGS4 from the similarity of the RGS domain RGS was that such an exhibit with other RGS family members. the specificity of for RGS4 by were developed with specificity for RGS7 in to RGS proteins and to provide a for expression endogenous RGS7 expression has been in tissue and cultured cells S. J. 1999; PubMed Scopus Google Scholar, Chen J. Neurosci. 2000; PubMed Scopus Google Scholar, A.M. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. D.S. Wang Q. Levay K. Slepak R. J. Neurosci. 2002; PubMed Scopus Google antibodies were a sequence of as the antibodies were for this RGS protein we endogenous RGS4 by and for cell that the most RGS4 protein, a was to the level of RGS4 mRNA in cell signals were for rat PC12M and AtT-20 cells little or signal was from rat rat rat or not with the of PC12M and cells revealed with RGS4 that to in detection of endogenous RGS4 was the of expression on cell We that of PC12M A and or cells expressed levels of RGS4 of cell than lower cell By contrast, the of Gαi detected was by cell density The level of expression of of proteins was by of the with or the A and which promote and/or of PC12 cells in for the of the cell in RGS4 protein revealed that and cells revealed difference between the of cells the of the cell as by cell The of cells in of the cell for and of PC12M cells were and that the mRNA levels for RGS4 were increased for cells with cells that the regulation of cell expression of RGS4 of that the on was PC12M cells were transfected with to RGS4 or RGS7 and of the was by the with a plasmid protein to the of of transfected PC12M revealed that of was with RGS4 and with RGS7 A of was observed for endogenous The RGS7 caused of endogenous RGS7 protein expression but was without on endogenous that the were and the of the RGS4 detected by with was that endogenous RGS4 could be by we the localization of endogenous PC12M cells were by for of and membrane cytosolic proteins heterologously RGS4 endogenous RGS4 was in the of PC12M the where are to be The of RGS4 and Gαi in the in be for by plasma membrane that with and other relatively possibility is that of the RGS4 with the low localization of the protein as has been reported for heterologously tagged forms of the protein A.A. Chidiac P. 2003; PubMed Scopus Google Scholar, Fisher J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Endogenous of RGS4 and in and PC12 the of antibodies endogenous proteins, have on in to the level of expressed protein. The of mRNA for RGS proteins in brain has been by in J. Neurosci. PubMed Google Scholar). We brain for expression of the protein to with of brain were and with and of protein from were for the of RGS4 and RGS7 by RGS4 protein was detected in and with lower levels in and RGS7 was to RGS4 that was well in levels of protein detected by well with the reported differences for mRNA J. Neurosci. PubMed Google Scholar). We with RGS to the endogenous expression of RGS4 and RGS7 proteins in of PC12M cells and from We detected of RGS4 of protein from PC12M cells and only of protein from on the other was more PC12M protein and protein. (11.Muma N.A. Mariyappa R. Williams K. Lee J.M. Synapse. 2003; 47: PubMed Scopus Google RGS4 in brain by with an RGS4 from but for the or of RGS4 were The of a from and rat brain that is which to the specificity of the for with or rat We the of RGS4 to be sequence and as a of in of PC12 cells and We the from with known of RGS4 and to be to whereas was to than of RGS4 a exposure of to we to be about more than the that we the available that either RGS4 is expressed to levels in brain with and rat or that the is to endogenous of Endogenous RGS4 and RGS4 and RGS7 have been reported to be to degradation by the A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, T. T. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). the signal for RGS4 was increased when PC12M or cells were to a or A and By contrast, on the endogenous levels of Gαi or RGS7 We that the expression of RGS4 was by a high rate of and we of protein by RGS4 to more than RGS7 and was by the in about of the RGS4 PC12M cells were to for whereas the expression of RGS7 and Gαi was apparently for which the of the cells not We an in endogenous RGS4 protein as a of PC12 cell exposure to the inhibitor with an in GAP activity the known mammalian RGS proteins, only and R4 family members have been to accelerate the GTPase activity of GAP activity in the was in the from cells to to cells in GAP activity is to be in to the in the of RGS4 in the membrane from the cells to by this mRNA for or was in PC12M cells by or of the (also known as could not be detected by with an that could than of and are R4 family members that be based on to be for degradation A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). we could not in PC12M cells by with an could than of RGS not of Endogenous RGS relatively of RGS4 the of endogenous RGS4 and GAP activity in cells with to regulation of degradation as a means for cells to levels of RGS4 protein. Because RGS4 has the capacity to regulate and signaling Hepler J.R. Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google we that RGS4 levels be promptly in response to of or both of G proteins and constitute a of feedback regulation of We to changes in expression of RGS4 protein by exposure of cells to G protein such as for and for a or or of the E.M. T. PubMed Scopus Google or (an of G proteins that is on but not of for from or to or were conducted on and but changes in RGS4 protein expression were and signaling promote in PC12 cells. S. Biochem. 1998; PubMed Scopus Google reported that of PC12 cells with or decreased RGS4 mRNA by We not an of or on the expression of RGS4 protein in PC12M cells not we that for decreased RGS4 protein levels by with in RGS7 and Gαi that this in RGS4 protein with a in RGS4 mRNA and By contrast, levels of mRNA for and were unaffected. for and was not detected is that the in RGS4 expression promote activity and contribute to the by which this of G protein in of protein and of PC12 cells S. S. PubMed Scopus Google Scholar). We substantial differences between endogenous and heterologously RGS proteins, sites utilized for of protein, and to and A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google reported in to a more of was by in vitro We observed the in cell as a of transfection with a that By contrast, we that only the was expressed in tissue or cultured cells. we that cells in not typically the of We a substantial of endogenous RGS4 protein was with membrane of PC12M cells. RGS4 is for regulation of Gα and the for of RGS4 to the a that been from studies of heterologously RGS4 Blumer K.J. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, A.A. Chidiac P. 2003; PubMed Scopus Google Scholar). the other of G protein that Gα, dissociated from Gβγ, is from the plasma membrane J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). cytosolic proteins, such as the of endogenous RGS proteins detected in D.S. Wang Q. Levay K. Cabrera J.L. Chen J. Willars G.B. Slepak V.Z. J. Biol. Chem. 2000; 275: 24872-24880Abstract Full Text Full Text PDF PubMed Scopus (132) Google could serve to Gα from the the of Gα to the plasma of RGS proteins in may have from finding substantial of Gα subunits in the Gilman A.G. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). We the of endogenous RGS4 to be on the of We this to the of protein as by and A. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google for in vitro and The the in of a protein to the of its amino The of RGS4 is to which targets the protein for and degradation by the mammalian cell transfection experiments, we not this protein was of this when an was a amino acid the a protein of the was a that RGS7 is to degradation by the T. T. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google we not that this of degradation is a characteristic of endogenous RGS proteins. T. T. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google reported that heterologously RGS7 is to degradation by the of this increased the level of expression of the protein. By contrast, we that not expression of endogenous RGS7 in PC12M this is with the protein sequence with which is not a amino of protein that endogenous RGS7 was to proteolysis We the of endogenous RGS7 in PC12M cells to its obligate association with may be when RGS7 is as has been by Slepak and D.S. Wang Q. Levay K. Cabrera J.L. Chen J. Willars G.B. Slepak V.Z. J. Biol. Chem. 2000; 275: 24872-24880Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). We that the low level of expression of endogenous RGS4 is related to a high rate of degradation to of the protein. The of RGS4 detected was about lower than RGS7 in The levels of RGS4 and RGS7 were only and of protein in whereas such as and are of membrane protein J. Biol. Chem. Full Text PDF PubMed Google Scholar). in the abundance of RGS and G proteins is with RGS proteins in vitro Wilkie T.M. Gilman A.G. Full Text Full Text PDF PubMed Scopus Google Scholar). We that localization of RGS proteins within cells of the brain, in signaling E.M. Wilkie T.M. Annu. Rev. Biochem. 2000; 69: 795-827Crossref PubMed Scopus (929) Google may be a determinant of which of Gαi and Gαo be to regulation by the relatively small of RGS proteins. of of brain and cell we a between the of RGS4 mRNA and the of protein by we detected RGS4 mRNA and protein in PC12M and cells but little or mRNA and protein in or cells. we observed of expression of RGS4 mRNA and protein by cell density or exposure of cells to NGF, a of regulation of mRNA and protein is not a we or of with the levels of RGS4 mRNA and protein in the not in J. Neurosci. 2003; PubMed Scopus Google Scholar). to the of protein to in the of on the expression and of RGS proteins. is the level of RGS4 protein expression dependent on cell the rate of RGS4 degradation and/or its rate of the levels of endogenous is that regulation of the rate of protein degradation a for increased RGS4 protein of PC12M cells with the in increased RGS4 expression of cell density for cells not of cells to the level of RGS4 expression in cells. that the of regulation is in on not to be a in cell not differences between the of cells of the cell Because RGS4 mRNA and protein levels were and with and cell the most is that RGS4 expression as a of increased activity related to increased in to that cell the RGS protein, overcome cell by for a G expression of a and this RGS protein as a feedback regulator of the J. Biol. PubMed Scopus Google Scholar). Because we observed a high rate of RGS4 degradation increased GAP activity in cells to a we that an appropriate or G protein RGS4 protein degradation in PC12M cells. could provide the means to the level of RGS4 protein for as a feedback which be more than a on we were to to regulate proteolysis of RGS4 either by receptor or by of G proteins. Although that endogenous RGS4 protein levels in PC12M cells are not by G protein the possibility remains that degradation of RGS4 may be a that or other means that we have to We Gilman and Ross for and for in and and for and cell and are for