I3 Subunits Research Articles

HIV-1 Nef Impairs Heterotrimeric G-protein Signaling by Targeting Gαi2 for Degradation through Ubiquitination

The HIV Nef protein is an important pathogenic factor that modulates cell surface receptor trafficking and impairs cell motility, presumably by interfering at multiple steps with chemotactic receptor signaling. Here, we report that a dominant effect of Nef is to trigger AIP4 E3 ligase-mediated Gα(i2) ubiquitination, which leads to Gα(i2) endolysosomal sequestration and destruction. The loss of the Gα(i2) subunit was demonstrable in many cell types in the context of gene transfection, HIV infection, or Nef protein transduction. Nef directly interacts with Gα(i2) and ternary complexes containing AIP4, Nef, and Gα(i2) form. A substantial reversal of Gα(i2) loss and a partial recovery of impaired chemotaxis occurred following siRNA knockdown of AIP4 or NEDD4 or by inhibiting dynamin. The N-terminal myristoyl group, (62)EEEE(65) motif, and (72)PXXP(75) motif of Nef are critical for this effect to occur. Nef expression does not affect a Gq(i5) chimera where the five C-terminal residues of Gq are replaced with those of Gα(i2). Lysine at position 296 of Gα(i2) was identified as the critical determinant of Nef-induced degradation. By specifically degrading Gα(i2), Nef directly subverts leukocyte migration and homing. Impaired trafficking and homing of HIV Nef-expressing lymphocytes probably contributes to early immune dysfunction following HIV infection.

Participation of central G‐alpha i2‐subunit proteins in blood pressure regulation and in countering salt‐sensitive hypertension

These studies tested the hypothesis that central Gai/o protein pathways are involved in mediating the cardiovascular depressor responses to central a2‐adrenoceptor activation and in countering the development of hypertension to a high NaCl challenge.MethodsThe a2‐agonist guanabenz (50 μg) was administered by intracerebroventricular (i.c.v.) injection to conscious instrumented Sprague‐Dawley (SD) rats pre‐treated (24‐h) i.c.v. with saline or oligodeoxynucleotides (ODN) against Gai1,i2, i3 and ao subunits (25 μg/each). Mean arterial pressure (MAP) and heart rate were recorded and urine collected for 150‐min (10‐min periods). Separate animals were implanted with miniosmotic pumps delivering i.c.v. Gai2 ODN (25 μg/day) and maintained on normal 0.4% NaCl (NS) or high 8% NaCl (HS) chow for 21‐days prior to study.ResultsIn vehicle pretreated SD rats on NS, i.c.v. guanabenz elicited bradycardia, hypotension, diuresis and natriuresis. Selective central down‐regulation of Gai1, i2, i3 or Gao protein did not alter bradycardic or diuretic responses to i.c.v. guanabenz. Gai2 protein down‐regulation prevented the hypotension (peak δMAP [mmHg]; guanabenz, ‐19±2 vs. Gai2 ODN + guanabenz, ‐3±2, P<0.05) and natriuresis to guanabenz (peak δUNaV [ μeq/min]; guanabenz, 7.6±0.8 vs. Gai2 ODN + guanabenz, 2.2±1, P<0.05). When infused (21‐day) with i.c.v. Gai2 ODN, SD rats maintained on HS developed hypertension (MAP [mmHg]; NS + Gai2 ODN, 129±2 vs. HS + Gai2 ODN, 147±3, P<0.05).ConclusionCentral Gαi2 protein‐gated pathways mediate the hypotensive and natriuretic responses to i.c.v. guanabenz. Further, these data suggest a previously unknown novel role for central Gαi2 protein pathways in preventing the development of salt‐sensitive hypertension. DK43337, HL 071212, AHA 0855293E, P20 RR018766

Chlamydomonas Flagellar Outer Row Dynein Assembly Protein Oda7 Interacts with Both Outer Row and I1 Inner Row Dyneins

We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.

A recombinant transductor–effector system: In vitro study of G inhibitory protein (G-alpha-i1) direct activators

Mutations and altered functionality of the inhibitory subfamily of G proteins (Gi) are involved in pathological states. Compounds able to activate Gi in a receptor-independent manner would be useful to treat these pathological conditions. Aimed to study Gi direct activation we have reconstituted a recombinant transductor–effector complex cloning both the mammalian Gα i1 subunit and adenylate cyclase (AC). The myristoylation of Gα, fundamental for interaction with AC, was obtained in the procaryotic expression host Escherichia coli transformed with a single plasmid containing both the coding sequences for human Gα i1 and Saccharomyces cerevisiae myristoyl transferase. AC-V isoform was obtained by the expression of its cytosolic domains. A recent synthesized molecule, named BC5, was tested to evaluate its pharmacological profile in a Gi/AC cell-free complex model. In this functional transductor–effector system BC5 was able to activate Gi signalling, moreover providing a new tool to give a better insight into G-protein receptor-independent modulation.

The Apelin Receptor Is Coupled to Gi1 or Gi2 Protein and Is Differentially Desensitized by Apelin Fragments

The apelin receptor is a G protein-coupled receptor to which two ligand fragments, apelin-(65-77) and apelin-(42-77), can bind. To address the physiological significance of the existence of dual ligands for a single receptor, we first compared the ability of the apelin fragments to regulate intracellular effectors, to promote G protein coupling, and to desensitize the response in Chinese hamster ovary cells expressing the murine apelin receptor. We found that both apelin fragments inhibited adenylyl cyclase and increased the phosphorylation of ERK or Akt. Using stably transfected cells expressing a pertussis toxin-insensitive alpha(i) subunit, we demonstrated that each apelin fragment promoted coupling of the apelin receptor to either Galpha(i1) or Galpha(i2) but not to Galpha(i3). Although preincubation with each apelin fragment induced a desensitization at the level of the three effectors, preincubation with apelin-(42-77) also increased basal effector activity. In addition, a C-terminal deletion of the apelin receptor decreased the desensitization induced by apelin-(65-77) but did not alter the desensitization pattern induced by apelin-(42-77). Finally, in umbilical endothelial cells, which we have recently shown to express the apelin receptor, the Galpha(i1) and Galpha(i2) subunits are also expressed, ERK and Akt phosphorylation is desensitized after preincubation with apelin-(65-77), and basal levels of Akt phosphorylation are increased after preincubation with apelin-(42-77). In summary, apelin fragments regulate the same effectors, via the preferential coupling of the apelin receptor to G(i1) or G(i2), but they promote a differential desensitization pattern that may be central to their respective physiological roles.

The R6A-1 peptide binds to switch II of Gα i1 but is not a GDP-dissociation inhibitor

Heterotrimeric G-proteins are molecular switches that convert signals from membrane receptors into changes in intracellular physiology. Recently, several peptides that bind heterotrimeric G-protein α subunits have been isolated including the novel Gα i1 · GDP binding peptides R6A and KB-752. The R6A peptide and its minimized derivative R6A-1 interact with Gα i1 · GDP. Based on spectroscopic analysis of BODIPYFL-GTPγS binding to Gα i1, it has been reported that R6A-1 has guanine nucleotide dissociation inhibitor (GDI) activity against Gα i1 [W.W. Ja, R.W. Roberts, Biochemistry 43 (28) (2004) 9265–9275]. Using radioligand binding, we show that R6A-1 is not a GDI for Gα i1 subunits. Furthermore, we demonstrate that R6A-1 reduces the fluorescence quantum yield of the Gα i1–BODIPYFL-GTPγS complex, thus explaining the previously reported GDI activity as a fluorescence artifact. We further show that R6A-1 has significant sequence similarity to the guanine nucleotide exchange factor peptide KB-752 that binds to switch II of Gα i1. We use competitive binding analysis to show that R6A-1 also binds to switch II of Gα subunits.

G-protein alpha subunit interaction and guanine nucleotide dissociation inhibitor activity of the dual GoLoco motif protein PCP-2 (Purkinje cell protein-2)

Purkinje cell protein-2 (PCP-2; L7/GPSM4) is a GoLoco motif-containing protein that is specifically expressed in Purkinje and retinal ON bipolar cells. An alternative splice variant of PCP-2 has recently been isolated which contains two GoLoco motifs. Although the second GoLoco motif (GL2) of PCP-2 has been reported to interact with Gα-subunits, a complete biochemical analysis of each individual motif of PCP-2 has not been performed. We demonstrate that the first GoLoco motif (GL1) of PCP-2 is equipotent as a guanine nucleotide dissociation inhibitor (GDI) towards Gα i1 and Gα i2, while it has sevenfold lower GDI activity for Gα i3 and greater than 20-fold lower GDI activity against Gα o. In contrast we found PCP-2 GL2 to be essentially equipotent as a GDI for all Gα i subunits, but it had negligible activity toward Gα o. Using co-immunoprecipitation from COS-7 cells, we found that PCP-2 was only able to interact with Gα i1 but not Gα o nor Gα-subunits from other families (Gα s, Gα q, or Gα 12). Mutational analysis of a non-canonical residue (glycine 24) in human PCP-2 GL1 provided evidence for heterogeneity in mechanisms of Gα i interactions with GoLoco motifs. Collectively, the data demonstrate that PCP-2 is a comparatively weak GoLoco motif protein that exhibits highest affinity interactions and GDI activity toward Gα i1, Gα i2, and Gα i3 subunits.

D2 dopamine receptor activation of potassium channels is selectively decoupled by Gαi‐specific GoLoco motif peptides

The GoLoco motif is a short polypeptide sequence found in G-protein signaling regulators such as regulator of G-protein signaling proteins type 12 and 14 and activator of G-protein signaling protein type 3. A unique property of the GoLoco motifs from these three proteins is their preferential interaction with guanosine diphosphate (GDP)-bound Galpha(i1), Galpha(i3) and, sometimes, Galpha(i2) subunits over Galpha(o) subunits. This interaction prevents both spontaneous guanine nucleotide release and reassociation of Galpha(i)-GDP with Gbetagamma. We utilized this property of the GoLoco motif to examine dopamine (D2 and D3) and somatostatin receptor coupling to G-protein-regulated inwardly rectifying potassium (GIRK) channels in mouse AtT20 cells. GoLoco motif peptides had no effect on either basal channel activity or the initial responses to agonists, suggesting that the GoLoco motif cannot disrupt pre-formed G-protein heterotrimers. GoLoco motif peptides did, however, interfere with human D2((short)) receptor coupling to GIRK channels as demonstrated by the progressively diminished responses after repeated agonist application. This behavior is consistent with some form of compartmentalization of D2 receptors and GIRK channels such that Gbetagamma subunits, freed by local receptor activation and prevented from reforming a heterotrimeric complex, are not functionally constrained within the receptor-channel complex and thus are unable to exert a persistent activating effect. In contrast, GoLoco motif peptides had no effect on either D3 or somatostatin coupling to GIRK channels. Our results suggest that GoLoco motif-based peptides will be useful tools in examining the specificity of G-protein-coupled receptor-effector coupling.

Accelerated transition from the double-positive to single-positive thymocytes in G i2-deficient mice

Deletion of alpha i2 subunit of heterotrimeric G proteins induces a 2-4-fold increase in the proportions of CD4 and CD8 single-positive (SP) thymocytes as compared with wild-type littermates, but how G alpha i2 is involved in thymocyte development is unknown. To determine a role for G alpha i2 in a specific developmental stage of thymocyte differentiation, we studied the ontogeny of thymocytes in G alpha i2-deficient mice. Our data show that an accelerated transition from the double-positive (DP) to SP thymocytes, rather than impairment in thymic emigration, accounts for a high proportion of the SP thymocytes in the absence of G alpha i2. Lack of G alpha i2 greatly augmented a response of thymocytes to TCR-mediated stimulation, as evidenced by enhanced proliferation of the DP thymocytes upon ligation of the TCRs. The augmented response may be the reason behind the expedited transition from the DP to SP thymocytes in the animal. In accordance with this, effects of G alpha i2 deficiency on CD8 or CD4 SP thymocyte differentiation required engagement of the TCRs with either MHC class I or MHC class II molecule. The abnormal thymocyte development resulted in an increase in positive selection, altered usage of TCR Vbeta gene, aberrant development of CD4+ CD25+ T regulatory cells and untimely thymic involution, the contribution of which to colitis development in the animal is discussed. These findings reveal a previously unappreciated role for G alpha i2 protein in clonal selection and functionality of thymocytes.

Immunoassay for visualization of protein‐protein interactions on Ni‐nitrilotriacetate support: Example of a laboratory exercise with recombinant heterotrimeric Gαi2β1γ2 Tagged by Hexahistidine from sf9 Cells

We have developed an in vitro assay for following the interaction between the α(i2) subunit and β(1γ2) dimer from sf9 cells. This method is suitable for education purposes because it is easy, reliable, nonexpensive, can be applied for a big class of 20 students, and avoid the commonly used kinetic approach, which does not allow visualization of protein-protein interaction.

Effects of partial agonists and Mg2+ ions on the interaction of M2 muscarinic acetylcholine receptor and G protein Galpha i1 subunit in the M2-Galpha i1 fusion protein.

We have expressed a M(2)-Galpha(i1) fusion protein in insect cells, in which the G protein alpha(i1) subunit was fused with a mutant of the muscarinic receptor M(2) subtype without glycosylation sites and the central part of the third intracellular loop. The M(2)-Galpha(i1) fusion protein showed GTP-sensitive, high-affinity agonist binding. Displacement curves by GDP of [(35)S]GTPgammaS binding shifted to the right in the presence of muscarinic agonists. The extent of the shift was greater for full agonists (120-150 fold) than for partial agonists (25-35 fold), and virtually no shift was observed for antagonists. The affinity for GDP decreased with increasing MgCl(2) concentration in the presence of an agonist but was not affected by MgCl(2) in the presence of an antagonist. These results indicate that the apparent affinity for GDP of the M(2)-Galpha(i1) fusion protein bound to a ligand represents the efficacy of the given ligand, and that Mg(2+) is required for the agonist-bound M(2) to interact with Galpha(i1), reducing its affinity for GDP. We propose that the agonist-M(2)-Galpha(i1) complex represents the transition state for the GDP-GTP exchange reaction catalyzed by agonist-bound receptors, and that the complex has different affinities for GDP depending on the species of the ligand bound to M(2) receptors.

TCR-mediated hyper-responsiveness of autoimmune Galphai2(-/-) mice is an intrinsic naïve CD4(+) T cell disorder selective for the Galphai2 subunit.

Heterotrimeric Gi signaling regulates immune homeostasis, since autoimmunity occurs upon disruption of this pathway. However, the role of the lymphocyte-expressed Galphai subunits (Galphai2 and 3) on T cell activation and cytokine production is poorly understood. To examine this role, we studied T lymphocytes from mice deficient in the Galphai2 or Galphai3 subunits. Galphai2(-/-) but not Galphai3(-/-) splenocytes were hyper-responsive for IFN-gamma and IL-4 production following activation through the TCR. Galphai2(-/-) T cells had a relaxed costimulatory requirement for IL-2 secretion and proliferation compared to wild-type cells. Purified naïve Galphai2(-/-) T cells produced more IL-2 than naïve wild-type T cells following TCR activation, indicating that the hyper-responsive cytokine profile was not due to the expanded Galphai2(-/-) memory T cells, but involved an intrinsic T cell alteration. Cytokine hyper-responsiveness was not seen when purified Galphai2(-/-) T cells were stimulated with phorbol myristic acetate/ionomycin, localizing the alteration to a proximal TCR-specific signaling pathway. Galphai2(-/-) CD4(+) T cells were distinguished from wild-type or Galphai3(-/-) T cells by a globally augmented TCR-induced calcium response. These findings indicate that Galphai2(-/-) mice have an intrinsic CD4(+) T cell abnormality in TCR signaling which may be one cause of augmented T cell effector function and Galphai2(-/-) autoimmune susceptibility.

Identification of a G Protein-Coupled Receptor for 5-oxo-Eicosatetraenoic Acid

5-Oxo-eicosatetraenoic acid (5-oxo-ETE), a metabolite of arachidonic acid, is known to act as a chemoattractant for neutrophils and eosinophils and is thought to be involved in inflammation and allergy. We have screened novel G protein-coupled receptors (GPCRs) from the human genome sequence by computational search (Takeda et al. FEBS Lett., 52097-520101 (2002)), and identified a GPCR (hGPCR48) among them as a receptor for 5-oxo-ETE. The fusion protein of hGPCR48 with G protein α i1 subunit was found to be activated by 5-oxo-ETE, 5(S)-hydroperoxy-eicosatetraenoic acid (5(S)-HPETE), and arachidonic acid with EC 5 0 of 5.5 nM, 19 nM, and 1.4 μM, respectively, as was assessed by agonist-stimulated [ 3 5 S]GTPγS binding activity. This EC 5 0 value for 5-oxo-ETE was essentially the same as the reported values for its binding to plasma membranes of neutrophils and for its effect to induce the increase of the intracellular Ca 2 + ions and the chemotaxis of neutrophils and eosinophils. The expression of hGPCR48 in neutrophils was evidenced by RT-PCR. These results indicate that hGPCR48 is a receptor for 5-oxo-ETE that is responsible for 5-oxo-ETE-induced chemotaxis of neutrophils. In addition, hGPCR48 might exert other physiological functions, because RT-PCR indicated its expression in liver and kidney at high levels and in the central nervous system at low levels.

Functional coupling with Gα q and Gα i1 protein subunits promotes high-affinity agonist binding to the neurotensin receptor NTS-1 expressed in Escherichia coli

To analyze the coupling of Gα subunits to the rat neurotensin receptor NTS-1 (NTR), fusion proteins were expressed in Escherichia coli with various Gα subunits covalently linked to the receptor C-terminus. The presence of Gα q or Gα i/q, in which the six C-terminal residues of Gα i1 were replaced with those from Gα q, increased the percentage of receptors in the agonist high-affinity state. This effect was less pronounced for wild-type Gα i1 and not observed for Gα i/s. Functional coupling of neurotensin receptor to Gα was demonstrated by neurotensin-induced [ 35S]GTPγS binding for the Gα q, Gα i/q and Gα i1 subunits, but not for Gα i/s. Our results extend previous findings of the dual coupling of NTR to pertussis toxin-sensitive and -insensitive G-proteins in Chinese hamster ovary cells with preference for the latter.

Coupling of the Insulin-like Growth Factor-I Receptor Tyrosine Kinase to Gi2 in Human Intestinal Smooth Muscle: Gβγ-DEPENDENT MITOGEN-ACTIVATED PROTEIN KINASE ACTIVATION AND GROWTH

Endogenous insulin-like growth factor-1 (IGF-I) stimulates growth of cultured human intestinal smooth muscle by activating distinct mitogen-activated protein (MAP) kinase-dependent and phosphatidylinositol 3-kinase-dependent signaling pathways. In Rat1 and Balb/c3T3 fibroblasts and in neurons the IGF-I receptor is coupled to an inhibitory G protein, G(i), which mediates G(beta)gamma-dependent MAP kinase activation. The present study determined whether in normal human intestinal smooth muscle cells the IGF-I receptor activates a heterotrimeric G protein and the role of G protein activation in mediating IGF-I-induced growth. IGF-I elicited IGF-I receptor tyrosine phosphorylation, resulting in the specific activation of G(i2). G(beta)gamma subunits selectively mediated IGF-I-dependent MAP kinase activation; G(alpha)i2 subunits selectively mediated IGF-I-dependent inhibition of adenylyl cyclase activity. IGF-I-stimulated MAP kinase activation and growth were inhibited by pertussis toxin, an inhibitor of G(i)/G(o) activation. Cyclic AMP inhibits growth of human intestinal muscle cells. IGF-I inhibited both basal and forskolin-stimulated cAMP levels. This inhibition was attenuated in the presence of pertussis toxin. IGF-I stimulated phosphatidylinositol 3-kinase activation, in contrast to MAP kinase activation, occurred independently of G(i2) activation. These data suggest that IGF-I specifically activates G(i2), resulting in concurrent G(beta)gamma-dependent stimulation of MAP kinase activity and growth, and G(alpha)i2-dependent inhibition of cAMP levels resulting in disinhibition of cAMP-mediated growth suppression.

Partial cDNAs encoding G-protein α subunits in the rat vestibular periphery

To begin understanding what G-proteins are involved in signal transduction in the vestibular periphery, the expression of G α subunits in rat primary afferent neurons (Scarpa's ganglia) and end-organs was studied. Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers corresponding to two conserved regions of the G α protein coding sequence produced partial cDNAs encoding two distinct forms of G α s subunit (G α s2 and anove) G α s2 subunit,GenBank accession number AF1841510); and two forms of G α i2 subunits. A novel truncated form of G α i2 (designated G α i2(vest),Gen Bank accession number AF189020) was detected in the vestibular periphery. G α i2(vest) was also expressed in rat cerebellum and heart. The possible role of the identified G α protein cDNAs in the function of the vestibular periphery is discussed.

Domains in the 1alpha dynein heavy chain required for inner arm assembly and flagellar motility in Chlamydomonas.

Flagellar motility is generated by the activity of multiple dynein motors, but the specific role of each dynein heavy chain (Dhc) is largely unknown, and the mechanism by which the different Dhcs are targeted to their unique locations is also poorly understood. We report here the complete nucleotide sequence of the Chlamydomonas Dhc1 gene and the corresponding deduced amino acid sequence of the 1alpha Dhc of the I1 inner dynein arm. The 1alpha Dhc is similar to other axonemal Dhcs, but two additional phosphate binding motifs (P-loops) have been identified in the NH(2)- and COOH-terminal regions. Because mutations in Dhc1 result in motility defects and loss of the I1 inner arm, a series of Dhc1 transgenes were used to rescue the mutant phenotypes. Motile cotransformants that express either full-length or truncated 1alpha Dhcs were recovered. The truncated 1alpha Dhc fragments lacked the dynein motor domain, but still assembled with the 1beta Dhc and other I1 subunits into partially functional complexes at the correct axoneme location. Analysis of the transformants has identified the site of the 1alpha motor domain in the I1 structure and further revealed the role of the 1alpha Dhc in flagellar motility and phototactic behavior.

Density of Guanine Nucleotide-Binding Proteins in Platelets of Patients with Major Depression: Increased Abundance of the Gα i2 Subunit and Down-Regulation by Antidepressant Drug Treatment

The aim of this study was to quantitate the density of guanine nucleotide-binding (G) protein subunits (inhibitory Gα i, stimulatory Gα s, Gα q/11, and Gβ) in platelets of unipolar depressed patients to assess the status of these signal transduction proteins in depression and the effects of antidepressant drug treatment. Blood platelets were collected from 22 drug-free depressed patients and 22 age- and sex-matched healthy controls. The levels of the various G protein subunits were assessed by immunoblotting techniques. The immunoreactivity of Gα i2 was increased (41%) and that of Gα i3 decreased (25%) in platelets of depressed patients. The levels of other G protein subunits (Gα s, Gα q/11, Gβ) did not change significantly with respect to those of control subjects. Chronic administration of cyclic antidepressant drugs (citalopram, clomipramine, imipramine) decreased the immunoreactivity of the up-regulated Gα i2 protein (31%). Since platelet Gα i2 proteins mediate the signaling of α 2A-adrenoceptors, the higher expression of Gα i2 is in line with the existence of supersensitivity of these receptors in major depression.

Dopamine enhances somatostatin receptor-mediated inhibition of adenylate cyclase in rat striatum and hippocampus.

Although there is evidence that suggests that dopamine (DA) has stimulatory effects on somatostatinergic transmission, it is unknown to date if DA increases the activity of the somatostatin (SS) receptor-effector system in the rat brain. In this study, we evaluated the effects of the administration of DA and the DA D1-like (D1, D5) receptor antagonist SCH 23390 and the D2-like (D2, D3, D4) receptor antagonist spiperone on the SS receptor-adenylate cyclase (AC) system in the Sprague-Dawley rat striatum and hippocampus. An intracerebroventricular injection of DA (0.5 microgram/rat) increased the number of SS receptors and decreased their apparent affinity in the striatum and hippocampus 15 hr after its administration. The simultaneous administration of the DA receptor antagonists SCH 23390 (0.25 mg/kg, ip) and spiperone (0.1 mg/kg, ip) before DA injection partially prevented the DA-induced increase in SS binding. The administration of SCH 23390 plus spiperone alone produced a significant decrease in the number of SS receptors in both brain areas studied at 15 hr after injection, an effect that disappeared at 24 hr. The increased number of SS receptors in the DA-treated rats was associated with an increased capacity of SS to inhibit basal and forskolin (FK)-stimulated (AC) activity in the striatum and hippocampus at 15 hr after injection. This effect had disappeared at 24 hr. By contrast, basal and FK-stimulated enzyme activities were unaltered after DA injection. No significant changes in the levels of the alpha i (alpha i1 + alpha i2) subunits were found in DA-treated rats as compared with control rats. In addition, the immunodetection of the alpha i1 or alpha i2 subunits showed no significant changes in their levels in DA-treated rats when compared with controls. DA injection also induced an increase in SS-like immunoreactive content in the rat striatum but not hippocampus at 15 hr after administration and returned to control values at 24 hr. These results provide direct evidence of a functional linkage between the dopaminergic and somatostatinergic systems at the molecular level.

The G-protein G(i) regulates mitosis but not DNA synthesis in growth factor-activated fibroblasts: a role for the nuclear translocation of G(i).

GTP binding proteins, heterotrimeric molecules composed of alpha-, beta-, and gamma-subunits, are known to serve as transducers of information from seven-transmembrane receptors. Activation of G-proteins has been generally considered to involve subunit dissociation, with G(alpha) separating from G(betagamma). However, we have found a receptor activation of G(i) in proliferating cells that differs from these models and involves the subcellular translocation of the alpha-subunit from the cell periphery to the nucleus where G(i alpha) binds to chromatin for the duration of mitosis. This report describes the mechanism of G(i) activation in Swiss 3T3 cells in response to serum, thrombin, and epidermal growth factor, and describes a role for G(i2) in the cell cycle. Agonists were found to be unable to induce the physical dissociation of G(i2) subunits. The alpha- and beta-subunits of G(i2) could be coimmunoprecipitated with a G(i alpha) antibody from both the membrane and nuclear fractions of long-term activated cultures, showing that G(i alpha 2) and G(i beta) are induced to comigrate to the nucleus in response to growth factor receptor activation. G(i2) appears to be activated in part by a postreceptor signal that can be mimicked by protein kinase C activation; this signal may be responsible for the convergence of the signaling mechanisms of these distinct seven-transmembrane and tyrosine kinase receptors. We suggest that translocation of G(i alpha) to the nucleus induced by either thrombin or EGF may occur without subunit dissociation. Functional studies of the role of G(i) showed that pertussis toxin does not block DNA synthesis in Swiss 3T3 fibroblasts induced by serum or thrombin, but that cell proliferation is retarded to each. These results provide direct evidence for a novel mechanism of GTP binding protein activation and for an essential role of G(i) in the induction of cell division by a variety of growth factor receptors. G(i) can carry out this role in control of cellular proliferation through its translocation to the nucleus of mitotic cells.