Pleiotropic Phenotype of a Genomic Knock-In of an RGS-Insensitive G184S Gnai2 Allele

Abstract

Signal transduction via guanine nucleotide binding proteins (G proteins) is involved in cardiovascular, neural, endocrine, and immune cell function. Regulators of G protein signaling (RGS proteins) speed the turn-off of G protein signals and inhibit signal transduction, but the in vivo roles of RGS proteins remain poorly defined. To overcome the redundancy of RGS functions and reveal the total contribution of RGS regulation at the G␣ i2 subunit, we prepared a genomic knock-in of the RGS-insensitive G184S Gnai2 allele. The G␣ i2 G184S knock-in mice show a dramatic and complex phenotype affecting multiple organ systems (heart, myeloid, skeletal, and central nervous system). Both homozygotes and heterozygotes demonstrate reduced viability and decreased body weight. Other phenotypes include shortened long bones, a markedly enlarged spleen, elevated neutrophil counts, an enlarged heart, and behavioral hyperactivity. Heterozygous G␣ i2 ؉/G184S mice show some but not all of these abnormalities. Thus, loss of RGS actions at G␣ i2 produces a dramatic and pleiotropic phenotype which is more evident than the phenotype seen for individual RGS protein knockouts.Cell-cell communication is fundamental to the maintenance of homeostasis. The G protein-coupled receptor superfamily is arguably the most abundant and diverse protein family in cellular signaling and is tightly regulated. A novel family of Ͼ20 proteins termed regulators of G protein signaling, or RGS proteins, both tonically inhibit G protein function and also serve as signal control points (2,22,34,39,69). RGS-mediated inhibition of G protein signaling occurs through direct binding of the RGS protein to the G␣ subunit, with subsequent GTPase-accelerating protein (GAP) actions to rapidly deactivate G␣ (2). Deactivation may be accelerated up to 1,000-fold and shuts down both G␣ and G␤␥ signals (42, 48). RGS proteins may also competitively inhibit G␣ binding to effectors such as phospholipase C (32). Most of the currently known RGS proteins interact with either Gi or Gq family G proteins and influence cyclic AMP (cAMP), Ca 2ϩ , mitogen-activated protein kinase, and ion channel signaling. There is strong evidence implicating them in the subsecond kinetics of G i -and G o -mediated ion channel activation and deactivation in the heart (10, 21, 36) and neurons (36). In addition, the conserved RGS domain has been found to serve as a multifunctional protein adapter which can recruit many effectors or regulators to the vicinity of activated G proteins (31,53,62). Notable examples include p115rhoGEF (30, 40) and GRK2 (44). There is also emerging interest in RGS proteins as drug targets (9,20,53,72).However, the physiological functions of RGS proteins remain poorly defined. A number of RGS knockouts have been reported (for example, RGS1, -2, -4, and -9). The RGS9-1 knockout shows prolonged visual potentials (7), and RGS9-2 disruption results in markedly enhanced responses to drugs of abuse, such as cocaine, amphetamines, and opiates (56, 71). A human disorder, bradyopsia, with r...

RGS inhibition at Gα i2 selectively potentiates 5-HT1A–mediated antidepressant effects

“…However, heterozygote Gαi2 (G184S/+) animals with partial RGS insensitivity at Gαi2 do not show similar physiological abnormalities (12), yet they exhibit this 5-HT1A-dependent antidepressant phenotype and improved sensitivity to serotonin-related antidepressants. These observations are consistent with previous biochemical studies showing a semidominant effect of this heterozygous gainof-function mutation (12) and suggest that endogenous RGS proteins play a major role in regulating the antidepressant/anxiolytic effects of endogenous serotonin and serotonergic agents acting at 5-HT1A receptors.…”

“…1B), homozygous [Gαi2 (G184S/G184S) ] and heterozygous [Gαi2 (G184S/+) ] RGS-insensitive mice also exhibited decreased immobility times (164 ± 10 and 183 ± 8 s, respectively) compared with wild-type animals (223 ± 9 s). Previous studies with Gαi2 (G184S/G184S) mice showed an increase in generalized activity measured over an 8-d period in the home cage (12). Despite this, we observed no differences between genotypes in measures of acute spontaneous activity in a new environment (Fig.…”

“…sensitivity at Gαi2 have a complex phenotype with abnormalities including low birth rate and low body weight (12). However, heterozygote Gαi2 (G184S/+) animals with partial RGS insensitivity at Gαi2 do not show similar physiological abnormalities (12), yet they exhibit this 5-HT1A-dependent antidepressant phenotype and improved sensitivity to serotonin-related antidepressants.…”

“…Wild-type Gαi2 (+/+) , heterozygous Gαi2 (G184S/+) , and homozygous Gαi2 (G184S/G184S) RGS-insensitive Gαi2 knock-in animals were derived from heterozygous breeding as previously described (12). All animals were backcrossed into the C57BL/6J strain for four generations and were matched for age and gender.…”

“…However, because of the large number of RGS proteins, defining the regulatory contribution of each has proven difficult, in part because of functional redundancy. To examine the combined regulatory role of endogenous RGS proteins, we used a knock-in mouse in which the GAP effects of endogenous RGS proteins to Gαi2 are silenced by a point mutation in the switch I region of Gαi2 (G184S) that disrupts its association with RGS proteins (11,12). Thus, all GAP-related RGS protein regulation is prevented without altering intrinsic functions of Gα, such as receptor interaction, association with βγ subunits, or coupling to downstream effectors (13).…”