Janet D. Robishaw

Heterotrimeric G-protein βγ-dimers in growth and differentiation

Heterotrimeric G-proteins are components of the signal transduction pathways for the soluble and cell-contact signals that regulate normal growth and differentiation. There is now a greater appreciation of the role of the Gβγ-dimer in the regulation of a variety of intracellular effectors, including ion channels, adenylyl cyclase, and phospholipase Cβ. In many cases, Gβγ-dimers are required for the activation of mitogen activated protein kinase (MAPK) pathways that promote cellular proliferation, although the underlying mechanisms have yet to be fully elucidated. Activation of phosphotidylinositol-3-kinase (PI3K) is a critical step in the intracellular transduction of survival signals. Gβγ-dimers directly activate PI3Kγ as well as the more widely distributed PI3Kβ. The activation of PI3Kγ by Gβγ-dimers likely involves direct binding of specific Gβγ-dimers to both subunits of PI3Kγ. Thus, Gβγ-dimers transmit signals from numerous receptors to a variety of intracellular effectors in distinct cellular contexts. Five distinct Gβ-subunits and 12 distinct Gγ-subunits have been identified. New experimental approaches are needed to elucidate the specific roles of individual Gβγ-dimers in the pathways that transduce signals for proliferation and survival.

Isolation of cDNA Clones Encoding Eight Different Human G Protein γ Subunits, Including Three Novel Forms Designated the γ4, γ10, and γ11 Subunits

With the growing awareness that the G protein β and γ subunits directly regulate the activities of various enzymes and ion channels, the importance of identifying and characterizing these subunits is underscored. In this paper, we report the isolation of cDNA clones encoding eight different human γ subunits, including three novel forms designated γ4, γ10, and γ11. The predicted protein sequence of γ4 shares the most identity (60-77%) with γ2, γ3, and γ7 and the least identity (38%) with γ1. The γ4 is modified by a geranylgeranyl group and is capable of interacting with both β1 and β2 but not with β3. The predicted protein sequence of γ10 shows only modest to low identity (35-53%) with the other known γ subunits, with most of the differences concentrated in the N-terminal region, suggesting γ10 may interact with a unique subclass of α. The γ10 is modified by a geranylgeranyl group and is capable of interacting with β1 and β2 but not with β3. Finally, the predicted protein sequence of γ11 shows the most identity to γ1 (76% identity) and the least identity to the other known γ (33-44%). Unlike most of the other known γ subunits, γ11 is modified by a farnesyl group and is not capable of interacting with β2. The close resemblance of γ11 to γ1 raises intriguing questions regarding its function since the mRNA for γ11 is abundantly expressed in all tissues tested except for brain, whereas the mRNA for γ1 is expressed only in the retina where the protein functions in phototransduction.

Subunit expression of signal transducing G proteins in cardiac tissue: Implications for phospholipase C-β regulation

In the heart, alpha-adrenergic, angiotensin II and endothelin signaling pathways modulate short-term changes in chronotropy and inotropy, and participate in the long-term control of cardiac growth. A shared feature of these signaling pathways is the stimulation of phosphatidylinositol (PI) turnover, which is thought to occur via G protein-mediated regulation of phospholipase C (PLC) activity. However, G protein subunits capable of regulating PLC activity have not been identified in different regions and cell types of the heart and members of the G protein-regulated PLC-beta isozyme family have not been documented in the heart. Using a battery of antipeptide specific antisera directed against the G protein alpha q, beta and gamma subunit families and against members of the PLC-beta, PLC-gamma and PLC-delta families, we demonstrated that heart tissues express the G protein alpha subunits alpha q and alpha 11, multiple G protein beta and gamma subunits, and PLC-beta 3, a phospholipase C isozyme regulated by either G protein alpha or beta gamma subunits. The degree of expression and distribution of these subunits differed between regions of the heart (atria versus ventricle) and changed with development. These data lay the ground work for future studies to determine the functional coupling of specific subsets of these components involved in receptor activation of PI turnover in the heart.

Differential Dependence of the D1 and D5 Dopamine Receptors on the G Protein γ7 Subunit for Activation of Adenylylcyclase

The D1 dopamine receptor, G protein γ7 subunit, and adenylylcyclase are selectively expressed in the striatum, suggesting their potential interaction in a common signaling pathway. To evaluate this possibility, a ribozyme strategy was used to suppress the expression of the G protein γ7 subunit in HEK 293 cells stably expressing the human D1 dopamine receptor. Prior in vitro analysis revealed that the γ7 ribozyme possessed cleavage activity directed exclusively toward the γ7 RNA transcript (Wang, Q., Mullah, B., Hansen, C., Asundi, J., and Robishaw, J. D. (1997)J. Biol. Chem. 272, 26040–26048). In vivoanalysis of cells transfected with the γ7 ribozyme showed a specific reduction in the expression of the γ7 protein. Coincident with the loss of the γ7 protein, there was a noticeable reduction in the expression of the β1 protein, confirming their interaction in these cells. Finally, functional analysis of ribozyme-mediated suppression of the β1 and γ7 proteins revealed a significant attenuation of SKF81297-stimulated adenylylcyclase activity in D1 dopamine receptor-expressing cells. By contrast, ribozyme-mediated suppression of the β1 and γ7 proteins showed no reduction of SKF81297-stimulated adenylylcyclase activity in D5 dopamine receptor-expressing cells. Taken together, these data indicate that the structurally related D1 and D5 dopamine receptor subtypes utilize G proteins composed of distinct βγ subunits to stimulate adenylylcyclase in HEK 293 cells. Underscoring the physiological relevance of these findings, single cell reverse transcriptase-polymerase chain reaction analysis revealed that the D1 dopamine receptor and the G protein γ7 subunit are coordinately expressed in substance P containing neurons in rat striatum, suggesting that the G protein γ7 subunit may be a new target for drugs to selectively alter dopaminergic signaling within the brain.

Mice with Deficiency of G Protein γ3 Are Lean and Have Seizures

ABSTRACT Emerging evidence suggests that the γ subunit composition of an individual G protein contributes to the specificity of the hundreds of known receptor signaling pathways. Among the twelve γ subtypes, γ3 is abundantly and widely expressed in the brain. To identify specific functions and associations for γ3, a gene-targeting approach was used to produce mice lacking the Gng3 gene (Gng3−/−). Confirming the efficacy and specificity of gene targeting, Gng3 −/− mice show no detectable expression of the Gng3 gene, but expression of the divergently transcribed Bscl2 gene is not affected. Suggesting unique roles for γ3 in the brain, Gng3 −/− mice display increased susceptibility to seizures, reduced body weights, and decreased adiposity compared to their wild-type littermates. Predicting possible associations for γ3, these phenotypic changes are associated with significant reductions in β2 and αi3 subunit levels in certain regions of the brain. The finding that the Gng3 −/− mice and the previously reported Gng7 −/− mice display distinct phenotypes and different αβγ subunit associations supports the notion that even closely related γ subtypes, such as γ3 and γ7, perform unique functions in the context of the organism.

Adenosine A2a receptor signaling and Golf assembly show a specific requirement for the γ7 subtype in the striatum

The adenosine A2A receptor (A2AR) is increasingly recognized as a novel therapeutic target in Parkinson disease. In striatopallidal neurons, the G-protein αolf subtype is required to couple this receptor to adenylyl cyclase activation. It is now well established that the βγ dimer also performs an active role in this signal transduction process. In principal, sixty distinct βγ dimers could arise from combinatorial association of the five known β and 12 γ subunit genes. However, key questions regarding which βγ subunit combinations exist and whether they perform specific signaling roles in the context of the organism remain to be answered. To explore these questions, we used a gene targeting approach to specifically ablate the G-protein γ7 subtype. Revealing a potentially new signaling paradigm, we show that the level of the γ7 protein controls the hierarchial assembly of a specific G-protein αolfβ2γ7 heterotrimer in the striatum. Providing a probable basis for the selectivity of receptor signaling, we further demonstrate that loss of this specific G-protein heterotrimer leads to reduced A2AR activation of adenylyl cyclase. Finally, substantiating an important role for this signaling pathway in pyschostimulant responsiveness, we show that mice lacking the G-protein γ7 subtype exhibit an attenuated behavioral response to caffeine. Collectively, these results further support the A2AR G-protein αolfβ2γ7 interface as a possible therapeutic target for Parkinson disease.

Alpha-1 adrenergic signaling in a cardiac murine atrial myocyte (HL-1) cell line

Activation of alpha-1 adrenergic receptors in the heart has been shown to result in increased contractile activity, cardiac fetal gene re-expression, and myocyte hypertrophy. Three alpha-1 adrenergic receptors have been identified through molecular cloning. Due to the limited selectivities of the currently available alpha-1 adrenergic receptor antagonists, the signaling pathways activated by specific subtypes in the heart remain unresolved. To resolve this dilemma, we have used a molecular approach to identify the signaling pathways and downstream genes that are engaged in response to activation of individual alpha-1 adrenergic subtypes in cardiac cells. We have transfected constitutively active alpha-1 adrenergic receptors (α1a-S290/293-AR [1] or the α1b-S288/294-AR [2]) subtypes into the cardiac murine myocyte cell line (HL-1) and studied the signal transduction pathway(s) and cardiac gene(s) activated by them. In this study, we demonstrate that the α1a-S290/293-AR [1] subtype preferentially couples to cardiac-specific atrial natriuretic factor (ANF) gene expression, while the α1b-S288/294-AR preferentially couples to activation of mitogen-activated protein kinase (MAPK), Ets-like transcription factor-1 (Elk1) and serum response element (SRE) signaling pathways. Endogenous alpha-1 adrenergic receptors are expressed, and stimulate phosphatidylinositol-hydrolysis upon activation with the alpha-1 agonist, phenylephrine.

Mice lacking the G protein γ3-subunit show resistance to opioids and diet induced obesity

Contributing to the obesity epidemic, there is increasing evidence that overconsumption of high-fat foods may be analogous to drug addiction in that the palatability of these foods is associated with activation of specific reward pathways in the brain. With this perspective, we report that mice lacking the G protein gamma(3)-subunit (Gng3(-/-) mice) show resistance to high-fat diet-induced weight gain over the course of a 12-wk study. Compared with Gng3(+/+) controls, female Gng3(-/-) mice exhibit a 40% reduction in weight gain and a 53% decrease in fat pad mass, whereas male Gng3(-/-) mice display an 18% reduction in weight gain and no significant decrease in fat pad mass. The basis for the lowered weight gain is related to reduced food consumption for female and male Gng3(-/-) mice of 13% and 14%, respectively. Female Gng3(-/-) mice also show a lesser preference for high-fat chow than their female Gng3(+/+) littermates, suggesting an attenuated effect on a reward pathway associated with overconsumption of fat. One possible candidate is the micro-opioid receptor (Oprm1) signaling cascade. Supporting a defect in this signaling pathway, Gng3(-/-) mice show marked reductions in both acute and chronic morphine responsiveness, as well as increases in endogenous opioid mRNA levels in reward-related regions of the brain. Taken together, these data suggest that the decreased weight gain of Gng3(-/-) mice may be related to a reduced rewarding effect of the high-fat diet resulting from a defect in Oprm1 signaling and loss of the G protein gamma(3)-subunit.

G-protein alpha subunit Gi(alpha)2 mediates erythropoietin signal transduction in human erythroid precursors.

Erythropoietin induces a dose-dependent increase in cytosolic calcium in human erythroblasts that is mediated by a voltage-independent Ca2+ channel. Inhibition of this response to erythropoietin by pertussis toxin suggests involvement of guanine nucleotide-binding regulatory proteins (G-proteins). The role of G-proteins in regulation of the erythropoietin-modulated Ca2+ channel was delineated here by microinjection of G-protein modulators or subunits into human erythroid precursors. This is the first report on the use of microinjection to study erythropoietin signal transduction in normal precursor cells. Fura-2 loaded day-10 burst-forming units-erythroid-derived erythroblasts were used for microinjection and free intracellular calcium concentration ([Ca(i)]) was measured with digital video imaging. BCECF (1,2,7-bis(2-carboxyethyl)-5-(and -6-)-carboxyfluorescein) was included in microinjectate, and an increase in BCECF fluorescence was evidence of successful microinjection. Cells were microinjected with nonhydrolyzable analogues of GTP, GTPgammaS or GDPbetaS, which maintain the alpha subunit in an activated or inactivated state, respectively. [Ca(i)] increased significantly in a dose-dependent manner after microinjection of GTPgammaS. However, injection of GDPbetaS blocked the erythropoietin-induced calcium increase, providing direct evidence that activation of a G-protein is required. To delineate which G-protein subunits are involved, alpha or betagamma transducin subunits were purified and microinjected as a sink for betagamma or alpha subunits in the erythroblast, respectively. Transducin betagamma, but not alpha, subunits eliminated the calcium response to erythropoietin, demonstrating the primary role of the alpha subunit. Microinjected antibodies to Gi(alpha)2, but not Gi(alpha)1 or Gi(alpha)3, blocked the erythropoietin-stimulated [Ca(i)] rise, identifying Gi(alpha)2 as the subunit involved. This was confirmed by the ability of microinjected recombinant myristoylated Gi(alpha)2, but not Gi(alpha)1 or Gi(alpha)3 subunits, to reconstitute the response of pertussis toxin-treated erythroblasts to erythropoietin. These data directly demonstrate a physiologic function of G-proteins in hematopoietic cells and show that Gi(alpha)2 is required in erythropoietin modulation of [Ca(i)] via influx through calcium channels.

Specificity of G protein alpha-gamma subunit interactions. N-terminal 15 amino acids of gamma subunit specifies interaction with alpha subunit.

The existence of multiple α, β, and subunits raises questions regarding the assembly of particular G proteins. Based on the results of a previous study (Rahmatullah, M., and Robishaw, J. D.(1994) J. Biol. Chem. 269, 3574-3580), we hypothesized that the assembly of G proteins may be determined by the interactions of the more structurally diverse α and subunits. This hypothesis was confirmed in the present study by showing striking differences in the abilities of the 1 and 2 subunits to interact with the the αo subunit. Chimeras of the 1 and 2 subunits were used to delineate which region is responsible. Support for the importance of the N-terminal region of the subunit comes from our observations that 1) the 2 subunit and the chimera bound strongly to the αo-agarose matrix, but the 1 subunit and the chimera bound weakly, if at all; 2) an N-terminal peptide made to the 2 subunit blocked the binding of the chimera to the αo-agarose matrix; 3) both the chimera and the N-terminal peptide were able to partially protect the αo subunit against tryptic cleavage; and 4) the chimera, but not the chimera, supported ADP-ribosylation of the αo subunit.