G protein betagamma complex translocation from plasma membrane to Golgi complex is influenced by receptor gamma subunit interaction

On activation of a receptor the G protein betagamma complex translocates away from the receptor on the plasma membrane to the Golgi complex. The rate of translocation is influenced by the type of gamma subunit associated with the G protein. Complementary approaches--imaging living cells expressing fluorescent protein tagged G proteins and assaying reconstituted receptors and G proteins in vitro--were used to identify mechanisms at the basis of the translocation process. Translocation of Gbetagamma containing mutant gamma subunits with altered prenyl moieties showed that the differences in the prenyl moieties were not sufficient to explain the differential effects of geranylgeranylated gamma5 and farnesylated gamma11 on the translocation process. The translocation properties of Gbetagamma were altered dramatically by mutating the C terminal tail region of the gamma subunit. The translocation characteristics of these mutants suggest that after receptor activation, Gbetagamma retains contact with a receptor through the gamma subunit C terminal domain and that differential interaction of the activated receptor with this domain controls Gbetagamma translocation from the plasma membrane.     

Structural insights into adhesion GPCR ADGRL3 activation and Gq,Gs, Gi,and G12 coupling

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The G protein beta subunit is a determinant in the coupling of Gs to the beta 1-adrenergic and A2a adenosine receptors

The signaling specificity of five purified G protein betagamma dimers, beta(1)gamma(2), beta(2)gamma(2), beta(3)gamma(2), beta(4)gamma(2), and beta(5)gamma(2), was explored by reconstituting them with G(s) alpha and receptors or effectors in the adenylyl cyclase cascade. The ability of the five betagamma dimers to support receptor-alpha-betagamma interactions was examined using membranes expressing the beta(1)-adrenergic or A2a adenosine receptors. These receptors discriminated among the defined heterotrimers based solely on the beta isoform. The beta(4)gamma(2) dimer demonstrated the highest coupling efficiency to either receptor. The beta(5)gamma(2) dimer coupled poorly to each receptor, with EC(50) values 40-200-fold higher than those observed with beta(4)gamma(2). Strikingly, whereas the EC(50) of the beta(1)gamma(2) dimer at the beta(1)-adrenergic receptor was similar to beta(4)gamma(2), its EC(50) was 20-fold higher at the A2a adenosine receptor. Inhibition of adenylyl cyclase type I (AC1) and stimulation of type II (AC2) by the betagamma dimers were measured. betagamma dimers containing Gbeta(1-4) were able to stimulate AC2 similarly, and beta(5)gamma(2) was much less potent. beta(1)gamma(2), beta(2)gamma(2), and beta(4)gamma(2) inhibited AC1 equally; beta(3)gamma(2) was 10-fold less effective, and beta(5)gamma(2) had no effect. These data argue that the beta isoform in the betagamma dimer can determine the specificity of signaling at both receptors and effectors.     

Receptor-mediated reversible translocation of the G protein betagamma complex from the plasma membrane to the Golgi complex

Heterotrimeric G proteins have been thought to function on the plasma membrane after activation by transmembrane receptors. Here we show that, after activation by receptors, the G protein betagamma complex selectively translocates to the Golgi. Receptor inactivation results in Gbetagamma translocating back to the plasma membrane. Both translocation processes occur rapidly within seconds. The efficiency of translocation is influenced by the type of gamma subunit present in the G protein. Distinctly different receptor types are capable of inducing the translocation. Receptor-mediated translocation of Gbetagamma can spatially segregate G protein signaling activity.     

One-step affinity purification of the G protein betagamma subunits from bovine brain using a histidine-tagged G protein alpha subunit

An efficient one-step affinity purification of bovine brain G protein betagamma subunits (betagamma's) is described. The betagamma's, in a detergent extract of brain membranes, are first dissociated from the alpha subunits (alpha's), reassociated with decahistidine-tagged alphail produced in bacteria, and then adsorbed onto Ni2+-nitrilotriacetic acid-agarose via the histidine tag. This mild adsorption retained the high activity of the ligand alpha's, in contrast to the commonly used chemical crosslinking methods. A wash step with a buffer containing chaotropic ions (SCN-) completely removed contaminating proteins that were refractory to washes with high concentrations of detergents, after which the highly purified betagamma's were eluted with a buffer containing Al3+, Mg2+, and F- ions. The obtained betagamma's were found to be fully functional, as assessed by their ability to support pertussis toxin-catalyzed ADP-ribosylation of alphail. Since the combination of the mild adsorption via the histidine tag and the wash with chaotropic ions can be easily applied to purifying betagamma's from various animal tissues, this new chromatographic method is expected to facilitate the purification of other membrane proteins that bind to Galpha and/or Galphabetagamma.     

Membrane translocation of P-Rex1 is mediated by G protein betagamma subunits and phosphoinositide 3-kinase

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac that is directly activated by the betagamma subunits of heterotrimeric G proteins and by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), which is generated by phosphoinositide 3-kinase (PI3K). Gbetagamma subunits and PIP(3) are membrane-bound, whereas the intracellular localization of P-Rex1 in basal cells is cytosolic. Activation of PI3K alone is not sufficient to promote significant membrane translocation of P-Rex1. Here we investigated the subcellular localization of P-Rex1 by fractionation of Sf9 cells co-expressing P-Rex1 with Gbetagamma and/or PI3K. In basal, serum-starved cells, P-Rex1 was mainly cytosolic, but 7% of the total was present in the 117,000 x g membrane fraction. Co-expression of P-Rex1 with either Gbetagamma or PI3K caused only an insignificant increase in P-Rex1 membrane localization, whereas Gbetagamma and PI3K together synergistically caused a robust increase in membrane-localized P-Rex1 to 23% of the total. PI3K-driven P-Rex1 membrane recruitment was wortmannin-sensitive. The use of P-Rex1 mutants showed that the isolated Dbl homology/pleckstrin homology domain tandem of P-Rex1 is sufficient for synergistic Gbetagamma- and PI3K-driven membrane localization; that the enzymatic GEF activity of P-Rex1 is not required for membrane translocation; and that the other domains of P-Rex1 (DEP, PDZ, and IP4P) contribute to keeping the enzyme localized in the cytosol of basal cells. In vitro Rac2-GEF activity assays showed that membrane-derived purified P-Rex1 has a higher basal activity than cytosol-derived P-Rex1, but both can be further activated by PIP(3) and Gbetagamma subunits.     

Stimulation of cellular signaling and G protein subunit dissociation by G protein betagamma subunit-binding peptides

We previously developed peptides that bind to G protein betagamma subunits and selectively block interactions between betagamma subunits and a subset of effectors in vitro (Scott, J. K., Huang, S. F., Gangadhar, B. P., Samoriski, G. M., Clapp, P., Gross, R. A., Taussig, R., and Smrcka, A. V. (2001) EMBO J. 20, 767-776). Here, we created cell-permeating versions of some of these peptides by N-terminal modification with either myristate or the cell permeation sequence from human immunodeficiency virus TAT protein. The myristoylated betagamma-binding peptide (mSIRK) applied to primary rat arterial smooth muscle cells caused rapid activation of extracellular signal-regulated kinase 1/2 in the absence of an agonist. This activation did not occur if the peptide lacked a myristate at the N terminus, if the peptide had a single point mutation to eliminate betagamma subunit binding, or if the cells stably expressed the C terminus of betaARK1. A human immunodeficiency virus TAT-modified peptide (TAT-SIRK) and a myristoylated version of a second peptide (mSCAR) that binds to the same site on betagamma subunits as mSIRK, also caused extracellular signal-regulated kinase activation. mSIRK also stimulated Jun N-terminal kinase phosphorylation, p38 mitogen-activated protein kinase phosphorylation, and phospholipase C activity and caused Ca2+ release from internal stores. When tested with purified G protein subunits in vitro, SIRK promoted alpha subunit dissociation from betagamma subunits without stimulating nucleotide exchange. These data suggest a novel mechanism by which selective betagamma-binding peptides can release G protein betagamma subunits from heterotrimers to stimulate G protein pathways in cells.     

Cooperation of Gq,Gi, and G12/13 in protein kinase D activation and phosphorylation induced by lysophosphatidic acid

To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Galpha(q/11) (Galpha(q/11) KO cells) or Galpha(12/13) (Galpha(12/13) KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control. In RK and Galpha(12/13) KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Galpha(12/13) KO cells in autophosphorylation activity) achieved at 3 microm. In contrast, LPA did not induce any significant increase in PKD activity in Galpha(q/11) KO cells. However, LPA induced a significantly increased PKD activity when Galpha(q/11) KO cells were transfected with Galpha(q). LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B. Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Galpha(12/13) KO cells. Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue. Our results indicate that G(q) is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to G(q), LPA requires additional G-protein pathways to elicit a maximal response with G(i) playing a critical role in Galpha(12/13) KO cells. We conclude that LPA induces PKD activation through G(q), G(i), and G(12) and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways.     

Tubulin binds specifically to the signal-transducing proteins, Gs alpha and Gi alpha 1

Participation of cytoskeletal elements in regulation of hormonal response and responsiveness has been suggested by several laboratories. Addition of dimeric tubulin to rat cerebral cortex synaptic membranes causes stable inhibition of adenylyl cyclase, and the molecular basis for this effect appears to require a direct interaction between tubulin and G proteins. To test whether such tubulin-G protein interaction occurred, several purified G proteins were bound to nitrocellulose, and 125I-tubulin overlay studies were performed. 125I-Tubulin bound to the alpha subunits of Gs and Gil with high specificity and an apparent Kd of approximately 130 nM. Other G protein alpha subunits (alpha i2, alpha i3, alpha 0, and transducin) displayed a much lower affinity for tubulin, despite the much closer relationship of those proteins to alpha il than to alpha s. Association of beta gamma subunits with alpha il or alpha s did not alter the binding of tubulin to these G protein heterotrimers, and the binding of a hydrolysis-resistant GTP analog to the alpha subunits was similarly without effect. These results suggest that tubulin forms complexes with specific G proteins and these complexes might provide a locus for the interaction of cytoskeletal components and signal transduction cascades. These results also provide evidence of a functional distinction among the closely related alpha i subtypes.     

Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.     

Thyrotropin-releasing hormone and gonadotropin-releasing hormone receptors activate phospholipase C by coupling to the guanosine triphosphate-binding proteins Gq and G11.

The regulation of pituitary hormone secretion by TRH and GnRH proceeds through similar mechanisms which employ phosphoinositide hydrolysis to generate intracellular signals. Proximal events involve receptor activation of heterotrimeric (alpha beta gamma) GTP-binding (G) proteins which regulate phospholipase (PLC) activity. Since TRH and GnRH actions are not affected by cholera or pertussis toxin, a novel G protein (Gp) was suggested to mediate receptor regulation. The required Gp protein has not been identified and this was the focus of the present study. Recent molecular cloning and biochemical studies have characterized two novel, pertussis toxin-insensitive alpha-subunit proteins of the Gq subfamily (alpha q and alpha 11) which regulate the activity of the beta 1 isoenzyme of PLC. Gq and G11 represent the best candidates for the PLC-activating G proteins which mediate the actions of TRH and GnRH. To test this directly, an antibody to the common Gq/11 alpha-subunit carboxyterminal sequence was generated and shown to react with unique 42-kilodalton Gq alpha and 43-kilodalton G11 alpha proteins in membranes from TRH-responsive GH3 cells and GnRH-responsive alpha T3-1 pituitary cells. The Gq/11 alpha peptide antibody was shown to immunodeplete the Gp activity of GH3 cell membrane extracts measured by reconstitution of the guanine nucleotide regulation of PLC-beta 1. In addition, the immunoglobulin G fraction of Gq/11 alpha peptide immune serum specifically inhibited TRH- and GnRH-stimulated PLC activity measured in the membranes of GH3 and alpha T3-1 cells, respectively. The results indicate that TRH and GnRH activation of PLC requires receptor coupling to a Gp protein(s) which corresponds to Gq, G11 or both.     

Endosome-to-Golgi transport is regulated by protein kinase A type II alpha

Studies of RII alpha-deficient B lymphoid cells and stable transfectants expressing the type II alpha regulatory subunit (RII alpha) of cAMP-dependent protein kinase (PKA), which is targeted to the Golgi-centrosomal area, reveal that the presence of a Golgi-associated pool of PKA type II alpha mediates a change in intracellular transport of the plant toxin ricin. The transport of ricin from endosomes to the Golgi apparatus, measured as sulfation of a modified ricin (ricin sulf-1), increased in RII alpha-expressing cells when PKA was activated. However, not only endosome-to-Golgi transport, but also retrograde ricin transport to the endoplasmic reticulum (ER), measured as sulfation and N-glycosylation of another modified ricin (ricin sulf-2), seemed to be increased in cells expressing RII alpha in the presence of a cAMP analog, 8-(4-chlorophenylthio)-cAMP. Thus, PKA type II alpha seems to be involved in both endosome-to-Golgi and Golgi-to-ER transport. Because ricin, after being retrogradely transported to the ER, is translocated to the cytosol, where it inhibits protein synthesis, we also investigated the influence of RII alpha expression on ricin toxicity. In agreement with the other data obtained, 8-(4-chlorophenylthio)-cAMP and RII alpha were found to sensitize cells to ricin, indicating an increased transport of ricin to the cytosol. In conclusion, our results demonstrate that transport of ricin from endosomes to the Golgi apparatus and further to the ER is regulated by PKA type II alpha isozyme.     

N-Myristoylation and betagamma play roles beyond anchorage in the palmitoylation of the G protein alpha(o) subunit

Many of the alpha subunits of heterotrimeric GTP-binding regulatory proteins (G proteins) are palmitoylated, a modification proposed to play a key role in the stable anchorage of the subunits to the plasma membrane. Palmitoylation of alpha subunits from the G(i) family is preceded by N-myristoylation, which alone or together with betagamma probably supports a reversible interaction of the alpha subunit with membrane as a prerequisite to the eventual incorporation of palmitate. Previous studies have not addressed, however, the question of whether membrane association alone, carried out through N-myristoylation, interaction with betagamma, or other events, is sufficient for palmitoylation. We report here for alpha(o) that it is not. We found that N-myristoylation is required for palmitoylation at least in part because it supports events subsequent to membrane attachment. Mutants of alpha(o) designed to target the subunit to membrane without an N-myristoyl group are unable to be palmitoylated as evaluated by incorporation of [(3)H]palmitate. Mutants of alpha(o) unable to interact normally with betagamma yet still attach to membrane demonstrate that betagamma, in contrast, is not required for palmitoylation. betagamma becomes necessary, however, when the N-myristoyl group is absent. Our results suggest that N-myristoylation and betagamma, while almost certainly relevant to the reversible interaction of alpha(o) with membrane, also play at least partly overlapping, post-anchorage roles in palmitoylation.     

Growth factor-induced cell division is paralleled by translocation of Gi alpha to the nucleus

Induction of mitosis by certain growth factors is inhibited by pertussis toxin, indicating that the GTP-binding protein, Gi, is involved in receptor signal transduction to initiate cell division. However, the substrates of receptor-activated Gi that are involved in mitosis have not been determined. The present study has examined whether Gi may directly modulate cell division by receptor-induced subcellular translocation of the alpha subunit of Gi (Gi alpha). Insulin and EGF, particularly when added together or in combination with phorbol dibutyrate (PdBu), induced a rapid (1-4 h) redistribution of Gi alpha from the plasma membrane to perinuclear sites in the cell. After 2 days of stimulation, Gi alpha had translocated into the nucleus of dividing cells and bound specifically to the separating chromatin of dividing nuclei. Unstimulated cells did not display translocation of Gi alpha. This demonstrates a direct involvement of Gi alpha in cell division, which provides an apparently uninterrupted link from growth factor receptor to nucleus.     

Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog.

Pseudohyphal differentiation, a filamentous growth form of the budding yeast Saccharomyces cerevisiae, is induced by nitrogen starvation. The mechanisms by which nitrogen limitation regulates this process are currently unknown. We have found that GPA2, one of the two heterotrimeric G protein alpha subunit homologs in yeast, regulates pseudohyphal differentiation. Deltagpa2/Deltagpa2 mutant strains have a defect in pseudohyphal growth. In contrast, a constitutively active allele of GPA2 stimulates filamentation, even on nitrogen-rich media. Moreover, a dominant negative GPA2 allele inhibits filamentation of wild-type strains. Several findings, including epistasis analysis and reporter gene studies, indicate that GPA2 does not regulate the MAP kinase cascade known to regulate filamentous growth. Previous studies have implicated GPA2 in the control of intracellular cAMP levels; we find that expression of the dominant RAS2(Gly19Val) mutant or exogenous cAMP suppresses the Deltagpa2 pseudohyphal defect. cAMP also stimulates filamentation in strains lacking the cAMP phosphodiesterase PDE2, even in the absence of nitrogen starvation. Our findings suggest that GPA2 is an element of the nitrogen sensing machinery that regulates pseudohyphal differentiation by modulating cAMP levels.     

PrKX is a novel catalytic subunit of the cAMP-dependent protein kinase regulated by the regulatory subunit type I

The human X chromosome-encoded protein kinase X (PrKX) belongs to the family of cAMP-dependent protein kinases. The catalytically active recombinant enzyme expressed in COS cells phosphorylates the heptapeptide Kemptide (LRRASLG) with a specific activity of 1.5 micromol/(min.mg). Using surface plasmon resonance, high affinity interactions were demonstrated with the regulatory subunit type I (RIalpha) of cAMP-dependent protein kinase (KD = 10 nM) and the heat-stable protein kinase inhibitor (KD = 15 nM), but not with the type II regulatory subunit (RIIalpha, KD = 2.3 microM) under physiological conditions. Kemptide and autophosphorylation activities of PrKX are strongly inhibited by the RIalpha subunit and by protein kinase inhibitor in vitro, but only weakly by the RIIalpha subunit. The inhibition by the RIalpha subunit is reversed by addition of nanomolar concentrations of cAMP (Ka = 40 nM), thus demonstrating that PrKX is a novel, type I cAMP-dependent protein kinase that is activated at lower cAMP concentrations than the holoenzyme with the Calpha subunit of cAMP-dependent protein kinase. Microinjection data clearly indicate that the type I R subunit but not type II binds to PrKX in vivo, preventing the translocation of PrKX to the nucleus in the absence of cAMP. The RIIalpha subunit is an excellent substrate for PrKX and is phosphorylated in vitro in a cAMP-independent manner. We discuss how PrKX can modulate the cAMP-mediated signal transduction pathway by preferential binding to the RIalpha subunit and by phosphorylating the RIIalpha subunit in the absence of cAMP.     

Human TREK2, a 2P domain mechano-sensitive K+ channel with multiple regulations by polyunsaturated fatty acids, lysophospholipids, and Gs, Gi, and Gq protein-coupled receptors

Mechano-sensitive and fatty acid-activated K(+) belong to the structural class of K(+) channel with two pore domains. Here, we report the isolation and the characterization of a novel member of this family. This channel, called TREK2, is closely related to TREK1 (78% of homology). Its gene is located on chromosome 14q31. TREK2 is abundantly expressed in pancreas and kidney and to a lower level in brain, testis, colon, and small intestine. In the central nervous system, TREK2 has a widespread distribution with the highest levels of expression in cerebellum, occipital lobe, putamen, and thalamus. In transfected cells, TREK2 produces rapidly activating and non-inactivating outward rectifier K(+) currents. The single-channel conductance is 100 picosiemens at +40 mV in 150 mm K(+). The currents can be strongly stimulated by polyunsaturated fatty acid such as arachidonic, docosahexaenoic, and linoleic acids and by lysophosphatidylcholine. The channel is also activated by acidification of the intracellular medium. TREK2 is blocked by application of intracellular cAMP. As with TREK1, TREK2 is activated by the volatile general anesthetics chloroform, halothane, and isoflurane and by the neuroprotective agent riluzole. TREK2 can be positively or negatively regulated by a variety of neurotransmitter receptors. Stimulation of the G(s)-coupled receptor 5HT4sR or the G(q)-coupled receptor mGluR1 inhibits channel activity, whereas activation of the G(i)-coupled receptor mGluR2 increases TREK2 currents. These multiple types of regulations suggest that TREK2 plays an important role as a target of neurotransmitter action.     

Conditional stimulation of type V and VI adenylyl cyclases by G protein betagamma subunits

In a yeast two-hybrid screen of mouse brain cDNA library, using the N-terminal region of human type V adenylyl cyclase (hACV) as bait, we identified G protein beta2 subunit as an interacting partner. Additional yeast two-hybrid assays showed that the Gbeta(1) subunit also interacts with the N-terminal segments of hACV and human type VI adenylyl cyclase (hACVI). In vitro adenylyl cyclase (AC) activity assays using membranes of Sf9 cells expressing hACV or hACVI showed that Gbetagamma subunits enhance the activity of these enzymes provided either Galpha(s) or forskolin is present. Deletion of residues 77-151, but not 1-76, in the N-terminal region of hACVI obliterated the ability of Gbetagamma subunits to conditionally stimulate the enzyme. Likewise, activities of the recombinant, engineered, soluble forms of ACV and ACVI, which lack the N termini, were not enhanced by Gbetagamma subunits. Transfection of the C terminus of G protein receptor kinase 2 to sequester endogenous Gbetagamma subunits attenuated the ability of isoproterenol to increase cAMP accumulation in COS-7 cells overexpressing hACVI even when G(i) was inactivated by pertussis toxin. Therefore, we conclude that the N termini of human hACV and hACVI are necessary for interactions with, and regulation by, Gbetagamma subunits both in vitro and in intact cells. Moreover, Gbetagamma subunits derived from a source(s) other than G(i) are necessary for the full activation of hACVI by isoproterenol in intact cells.     

Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the alpha subunits of Gq family.

High efficiency transient transfection of Cos-7 cells was previously used to establish the functional coupling between G alpha q/G alpha 11 and phospholipase C beta 1 (Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817). Here the same system was used to study the functional coupling between other guanine nucleotide-binding regulatory protein (G-protein) alpha subunits and phospholipases and to study which G alpha subunits mediate the activation of phospholipase C by the alpha 1-adrenergic receptor subtypes, alpha 1 A, alpha 1 B, and alpha 1 C. We found that G alpha 14 and G alpha 16 behaved like G alpha 11 or G alpha q, i.e. they could activate endogenous phospholipases in Cos-7 cells in the presence of AIFn. The synergistic increase in inositol phosphate release in Cos-7 cells after they were cotransfected with cDNAs encoding G alpha subunits and phospholipase C beta 1 indicates that both G alpha 16 and G alpha 14 can activate phospholipase C beta 1. The activation of phospholipase C beta 1 was restricted to members of the Gq subfamily of alpha subunits. They activated phospholipase C beta 1 but not phospholipase C gamma 1, gamma 2, or phospholipase C delta 3. The cotransfection of Cos-7 cells with cDNAs encoding three different alpha 1-adrenergic receptors and G alpha q or G alpha 11 leads to an increase in norepinephrine-dependent inositol phosphate release. This indicates that G alpha q or G alpha 11 can mediate the activation of phospholipase C by all three subtypes of alpha 1-adrenergic receptors. With the same assay system, G alpha 16 and G alpha 14 appear to be differentially involved in the activation of phospholipase C by the alpha 1-adrenergic receptors. The alpha 1 B subtype receptor gave a ligand-mediated synergistic response in the cells cotransfected with either G alpha 14 or G alpha 16. However, the alpha 1 C receptor responded in cells cotransfected with G alpha 14 but not G alpha 16, and the alpha 1 A receptor showed little synergistic response in cells transfected with either G alpha 14 or G alpha 16. The ability of the alpha 1 A and alpha 1 C receptors to activate phospholipase C through G alpha q and G alpha 11 was also demonstrated in a cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)