Comparison of mRNA Expression of Two Regulators of G-Protein Signaling,RGS1/BL34/1R20andRGS2/G0S8, in Cultured Human Blood Mononuclear Cells

Abstract

RGS1 and RGS2 are members of a new class of regulators of G-protein signaling identified by their selective mRNA expression either in phorbol ester (TPA)-stimulated human B lymphocytes (RGS1/1R20/BL34) or in blood mononuclear cells treated with the T-cell lectin concanavalin A (ConA) and cycloheximide (RGS2/G0S8). The RGS1 gene shows low basal mRNA expression in freshly purified blood mononuclear cells, which increases upon incubation for a day. In contrast, RGS2 initially shows high basal levels of mRNA expression, which subsequently decrease. Expression of both genes increases in response to ConA, with RGS2 mRNA levels increasing briskly to a maximum between 0.5 and 1 hr and decreasing to baseline by 6 hr, whereas the RGS1 mRNA increase is delayed reaching a maximum between 1 and 2 hr. RGS1 mRNA levels increase much more in response to a protein kinase C activator (TPA), than to a calcium ionophore (ionomycin), whereas the opposite is true for RGS2. We suggest that ConA elevates RGS2 on the basis of its ability to increase intracellular calcium, and that RGS2 may be involved in the regulation of intracellular calcium. The distinction between RGS1 and RGS2 is further emphasized by studies indicating that recombinant RGS2 does not bind in vitro to two members of the G(i) subfamily of G-protein alpha-subunits for which recombinant RGS1 has high affinity.

Regulation of T cell activation, anxiety, and male aggression by RGS2

“…2B). Consistent with the identification of RGS2 as an early response gene in activated T cells (13)(14), the proliferative defect was most obvious at 24 h of stimulation. Induction and kinetics of apoptosis were comparable among rgs2 ϩ/Ϫ and rgs2 Ϫ/Ϫ T cells (not shown).…”

“…The RGS family member rgs2 was cloned as an early response gene up-regulated in activated T cells (13)(14). RGS2 is also up-regulated in central nervous system (CNS) neurons after stimuli that evoke long-term neuronal plasticity (15)(16).…”

RGS Proteins Determine Signaling Specificity of Gq-coupled Receptors

“…Expression and Purification of RGS Proteins-Recombinant RGS1, RGS2, RGS4, and RGS16 proteins were tagged at the N terminus with the sequence MH 6 MG using the pET19b and a modified pQE60 expression vector, respectively (Qiagen), expressed in Escherichia coli and purified as described (10,14,15). Briefly, a 10-ml overnight culture grown at 37°C in T7 medium supplemented with 100 g/ml ampicillin and 1% glucose was used to inoculate 2 liter of T7/100 g/ml ampicillin medium at 30°C.…”

“…G␣ 11 Ϫ/Ϫ;G␣ 14 Ϫ/Ϫ double homozygous null mice were obtained from intercrossing the offspring of the single knockout mice. To produce G␣ 15 Ϫ/Ϫ knockout mice, integration of the G␣ 15 targeting vector replaced exons 3, 4, and a portion of 5 with PGK::Neo in the inverse orientation. G␣ 15 Ϫ/Ϫ mice are viable and fertile with no apparent behavioral or morphologic defects.…”

“…Recent work has identified over 20 RGS proteins expressed in mammals (12, 16, 18 -22). In vitro studies suggest that several mammalian RGS proteins, including RGS1, RGS4, and RGS16, have GAP activity toward different G i and/or G q class ␣ subunits but not G␣ s or G␣ 12 (10,11,15,(23)(24)(25). RGS4 inhibited G q -dependent PLC␤ activation in Xenopus oocytes and transfected COS and HEK293 cells (24 -27).…”