Differentially regulated expression of endogenous RGS4 and RGS7.
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.
Krumins
Andrejs M
AM
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041, USA.
Barker
Sheryll A
SA
Huang
Chunfa
C
Sunahara
Roger K
RK
Yu
Kan
K
Wilkie
Thomas M
TM
Gold
Stephen J
SJ
Mumby
Susanne M
SM
D000818:Animals; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000096:biosynthesis*; D005786:Gene Expression Regulation; D006801:Humans; D020710:RGS Proteins / Q000096:biosynthesis*; D051381:Rats; D015398:Signal Transduction
D020710:RGS Proteins; C492039:RGS7 protein, human; C115771:Rgs7 protein, rat; C098799:RGS4 protein; D019204:GTP-Binding Proteins