Protein Betagamma Subunits Research Articles

G protein {beta}{gamma} subunits mediate presynaptic inhibition of transmitter release from rat superior cervical ganglion neurones in culture.

The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic alpha2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of Galpha(i/o)betagamma subunits. Expression of Galpha transducin, an agent which sequesters G protein betagamma (Gbetagamma) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified Gbetagamma subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of Gbetagamma subunits. The Ca(2+) channel blocker Cd(2+) mimicked NA effects on transmitter release. Cd(2+), NA and Gbetagamma subunits also inhibited somatic Ca(2+) current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that Gbetagamma subunits functionally mediate inhibition of transmitter release by alpha2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.

Multiple actions of extracellular ATP and adenosine on calcium currents mediated by various purinoceptors in neurons of nucleus tractus solitarius.

Whole-cell patch-clamp recordings were performed on freshly dissociated nucleus tractus solitarius (NTS) of rat to determine the action of extracellular adenosine 5'-triphosphate (ATP) and adenosine (ADO) on voltage-dependent calcium channel (VDCC) currents (I(Ca)). Application of ATP and ATP-analog inhibited I(Ca). The rank order of potency of inhibition of I(Ca) was 2-methylthioATP (2-MeSATP) > ATP > adenosine 5'-diphosphate (ADP) >> alpha,beta-methylene ATP (alpha,beta-MeATP) = uridine 5'-triphosphate (UTP). Application of ADO receptor agonists also inhibited I(Ca). The rank order of potency of inhibition of I(Ca) was N(6)-cyclohexyladenosine (CHA) > ADO > 2-(4-(2-carboxyethyl)phenylethylamino)adenosine-5'-N-ethylcarboxamideadenosine (CGS-21680) > N(6)-2-(4-aminophenyl)ethyladenosine (APNEA). Application of prepulse attenuated these inhibition. Both intracellular dialysis of guanosin 5'-O-(2-thiodiphosphate) (GDP-beta-S) and anti-G(i) antibody also attenuated these inhibition. L-, N- and P/Q-type VDCCs were inhibited by ATP. In contrast, N- and P/Q-type VDCCs were inhibited by ADO. In addition to inhibition, application of 100 microM ATP facilitated I(Ca). Intracellular dialysis of GDP-beta-S did not attenuate these facilitations. In conclusion, activation of P2Y purinoceptors inhibits L-, N- and P/Q-types VDCCs via G(i)-protein betagamma subunits. Activation of A(1) and/or A(2) receptors inhibit N- and P/Q-types VDCCs via G(i)-protein betagamma subunits. Activation of P2X purinoceptors facilitates Ca(2+) entry in NTS.

Dominant-negative Inhibition of Pheromone Receptor Signaling by a Single Point Mutation in the G Protein α Subunit

In yeast, two different constitutive mutants of the G protein alpha subunit have been reported. Gpa1(Q323L) cannot hydrolyze GTP and permanently activates the pheromone response pathway. Gpa1(N388D) was also proposed to lack GTPase activity, yet it has an inhibitory effect on pheromone responsiveness. We have characterized this inhibitory mutant (designated Galpha(ND)) and found that it binds GTP, interacts with G protein betagamma subunits, and exhibits full GTPase activity in vitro. Although pheromone leads to dissociation of the receptor from wild-type G protein, the same treatment promotes stable association of the receptor with Galpha(ND). We conclude that agonist binding to the receptor promotes the formation of a nondissociable complex with Galpha(ND), and in this manner prevents activation of the endogenous wild-type G protein. Dominant-negative mutants may be useful in matching specific receptors and their cognate G proteins and in determining mechanisms of G protein signaling specificity.

Involvement of G protein betagamma-subunits in diverse signaling induced by G(i/o)-coupled receptors: study using the Xenopus oocyte expression system.

We studied the functions of betagamma-subunits of G(i/o) protein using the Xenopus oocyte expression system. Isoproterenol (ISO) elicited cAMP production and slowly activating Cl(-) currents in oocytes expressing beta(2)-adrenoceptor and the protein kinase A-dependent Cl(-) channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. 5-Hydroxytryptamine (5-HT), [d-Ala(2), d-Leu(5)]-enkephalin (DADLE), and baclofen enhanced ISO-induced cAMP levels and CFTR currents in oocytes expressing beta(2)-adrenoceptor-CFTR and 5-HT(1A) receptor (5-HT(1A)R), delta-opioid receptor, or GABA(B) receptor, respectively. 5-HT also enhanced pituitary adenylate cyclase activating peptide (PACAP) 38-induced cAMP levels and CFTR currents in oocytes expressing PACAP receptor, CFTR and 5-HT(1A)R. The 5-HT-induced enhancement of G(s)-coupled receptor-mediated currents was abrogated by pretreatment with pertussis toxin (PTX) and coexpression of G transducin alpha (G(t)alpha). The 5-HT-induced enhancement was further augmented by coexpression of the Gbetagamma-activated form of adenylate cyclase (AC) type II but not AC type III. Thus betagamma-subunits of G(i/o) protein contribute to the enhancement of G(s)-coupled receptor-mediated responses. 5-HT and DADLE did not elicit any currents in oocytes expressing 5-HT(1A)R or delta-opioid receptor alone. They elicited Ca(2+)-activated Cl(-) currents in oocytes coexpressing these receptors with the Gbetagamma-activated form of phospholipase C (PLC)-beta2 but not with PLC-beta1. These currents were inhibited by pretreatment with PTX and coexpression of G(t)alpha, suggesting that betagamma-subunits of G(i/o) protein activate PLC-beta2 and then cause intracellular Ca(2+) mobilization. Our results indicate that betagamma-subunits of G(i/o) protein participate in diverse intracellular signals, enhancement of G(s)-coupled receptor-mediated responses, and intracellular Ca(2+) mobilization.

Rap1-induced p38 Mitogen-activated Protein Kinase Activation Facilitates AMPA Receptor Trafficking via the GDI·Rab5 Complex: POTENTIAL ROLE IN (S)-3,5-DIHYDROXYPHENYLGLYCINE-INDUCED LONG TERM DEPRESSION

Recent evidence has emphasized the importance of p38 mitogen-activated protein kinase (MAPK) in the induction of metabotropic glutamate receptor (mGluR)-dependent long term depression (LTD) at hippocampal CA3-CA1 synapses. However, the cascade responsible of mGluR to activate p38 MAPK and the signaling pathway immediately downstream from it to induce synaptic depression is poorly understood. Here, we show that transient activation of group I mGluR with the selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) activates p38 MAPK through G protein betagamma-subunit, small GTPase Rap1, and MAPK kinase 3/6 (MKK3/6), thus resulting in mGluR5-dependent LTD. Furthermore, our data clearly show that an accelerating AMPA receptor endocytosis by stimulating the formation of guanyl nucleotide dissociation inhibitor-Rab5 complex is a potential downstream processing of p38 MAPK activation to mediate DHPG-LTD. These results suggest an important role for Rap1-MKK3/6-p38 MAPK pathway in the induction of mGluR-dependent LTD by directly coupling to receptor trafficking machineries to facilitate the loss of synaptic AMPA receptors.

Purification of recombinant G protein alpha subunits from Escherichia coli.

The purification of recombinant G protein a subunits expressed in Escherichia coli (E. coli) is a convenient and inexpensive method to obtain homogeneous preparations of protein for biochemical and biophysical analyses. Wild-type and mutant forms of G alpha are easily produced for analysis of their intrinsic biochemical properties, as well as for reconstitution with receptors, effectors, regulators, and G protein beta gamma subunits. Methods are described for the expression of Gi alpha and Gs alpha proteins in E. coli. Protocols are provided for the purification of untagged G protein a subunits using conventional chromatography and histidine (His)-tagged subunits using metal chelate chromatography. Modification of G alpha with myristate can be recapitulated in E. coli by expressing N-myristoyltransferase (NMT) with its G protein substrate. Protocols for the production and purification of myristoylated G alpha are presented.

Competitive and Synergistic Interactions of G Protein β2 and Ca2+ Channel β1b Subunits with Cav2.1 Channels, Revealed by Mammalian Two-hybrid and Fluorescence Resonance Energy Transfer Measurements

Presynaptic Ca2+ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein betagamma subunits modulate the binding of the Ca2+ channel beta subunit on the Ca2+ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca2+ channel beta and G protein beta subunits on the two separate binding sites, i.e. the loopI-II and the C terminus, and on the full-length P/Q-type alpha12.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca2+ channel beta1b subunit induces a strong fluorescent signal when interacting with the loopI-II but not with the C terminus. In contrast, the G protein beta subunit induces FRET signals on both the C terminus and loopI-II. Analysis of the interactions on the full-length channel indicates that Ca2+ channel beta1b and G protein beta subunits bind to the alpha1 subunit at the same time. Coexpression of the G protein increases the FRET signal between alpha1/beta1b FRET pairs but not for alpha1/beta1b FRET pairs where the C terminus was deleted from the alpha1 subunit. The results suggest that the G protein alters the orientation and/or association between the Ca2+ channel beta and alpha12.1 subunits, which involves the C terminus of the alpha1 subunit and may corresponds to a new conformational state of the channel.

Interaction of G protein beta subunit with inward rectifier K(+) channel Kir3.

G protein betagamma subunits bind and activate G protein-coupled inward rectifier K+ (GIRK) channels. This protein-protein interaction is crucial for slow hyperpolarizations of cardiac myocytes and neurons. The crystal structure of Gbeta shows a seven-bladed propeller with four beta strands in each blade. The Gbeta/Galpha interacting surface contains sites for activating GIRK channels. Furthermore, our recent investigation using chimeras between Gbeta1 and yeast beta (STE4) suggested that the outer strands of blades 1 and 2 of Gbeta1 could be an interaction area between Gbeta1 and GIRK. In this study, we made point mutations on suspected residues on these outer strands and investigated their ability to activate GIRK1/GIRK2 channels. Mutations at Thr-86, Thr-87, and Gly-131, all located on the loops between beta-strands, substantially reduced GIRK channel activation, suggesting that these residues are Gbeta/GIRK interaction sites. These mutations did not affect the expression of Gbeta1 or its ability to stimulate PLCbeta2. These residues are surface-accessible and located outside Gbeta/Galpha interaction sites. These results suggest that the residues on the outer surface of blades 1 and 2 are involved in the interaction of Gbetagamma with GIRK channels. Our study suggests a mechanism by which different effectors use different blades to achieve divergence of signaling. We also observed that substitution of alanine for Trp-332 of Gbeta1 impaired the functional interaction of Gbeta1 with GIRK, in agreement with the data on native neuronal GIRK channels. Trp-332 plays a critical role in the interaction of Gbeta1 with Galpha as well as all effectors so far tested.

G Protein betagamma subunits stimulate p114RhoGEF, a guanine nucleotide exchange factor for RhoA and Rac1: regulation of cell shape and reactive oxygen species production.

Rho GTPases integrate the intracellular signaling in a wide range of cellular processes. Activation of these G proteins is tightly controlled by a number of guanine nucleotide exchange factors (GEFs). In this study, we addressed the functional role of the recently identified p114RhoGEF in in vivo experiments. Activation of endogenous G protein-coupled receptors with lysophosphatidic acid resulted in activation of a transcription factor, serum response element (SRE), that was enhanced by p114RhoGEF. This stimulation was inhibited by the functional scavenger of Gbetagamma subunits, transducin. We have determined that Gbetagamma subunits but not Galpha subunits of heterotrimeric G proteins stimulated p114RhoGEF-dependent SRE activity. Using coimmunoprecipitation assay, we have determined that Gbetagamma subunits interacted with full-length and DH/PH domain of p114RhoGEF. Similarly, Gbetagamma subunits stimulated SRE activity induced by full-length and DH/PH domain of p114RhoGEF. Using in vivo pull-down assays and dominant-negative mutants of Rho GTPases, we have determined that p114RhoGEF activated RhoA and Rac1 but not Cdc42 proteins. Functional significance of RhoA activation was established by the ability of p114RhoGEF to induce actin stress fibers and cell rounding. Functional significance of Rac1 activation was established by the ability of p114RhoGEF to induce production of reactive oxygen species (ROS) followed by activation of NADPH oxidase enzyme complex. In summary, our data showed that the novel guanine nucleotide exchange factor p114RhoGEF regulates the activity of RhoA and Rac1, and that Gbetagamma subunits of heterotrimeric G proteins are activators of p114RhoGEF under physiological conditions. The findings help to explain the integrated effects of LPA and other G-protein receptor-coupled agonists on actin stress fiber formation, cell shape change, and ROS production.

Involvement of intramolecular interactions in the regulation of G protein-coupled receptor kinase 2.

The G protein-coupled receptor (GPCR) kinase GRK2 phosphorylates G protein-coupled receptors in an agonist-dependent manner. GRK2 activity is modulated through interactions of diverse domains of the kinase with G protein betagamma subunits, several lipids, anchoring proteins, and activated receptors. We report that kinase activity toward either GPCR (rhodopsin) or a synthetic peptide substrate is enhanced in the presence of GST-GRK2 fusion proteins or peptides corresponding to either N- or C-terminal sequences of GRK2. This direct stimulatory action of intrinsic domains on GRK2 activity does not add to the effect of other regulators, such as Gbetagamma subunits, and strongly suggests the existence of some mode of autoregulation. The existence of regulatory intramolecular interactions in GRK2 is supported by the facts that a C-terminal peptide protects the N-terminal region from proteolytic cleavage and that two domains of GRK2 independently coexpressed in cells associate as assessed by immunoprecipitation. Molecular modeling suggests that intramolecular interactions among the N-terminal, C-terminal and kinase domains would keep GRK2 in a constrained conformation characteristic of an inactive, basal state. Our model proposes that disruption of such intramolecular contacts by intermolecular interactions with regulatory proteins (mimicked by exogenously added kinase fragments in vitro) would promote the conformational changes required to bring about GRK2 translocation and activation.

Stimulation of Cellular Signaling and G Protein Subunit Dissociation by G Protein βγ 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.

Extracellular ATP-induced calcium channel inhibition mediated by P1/P2Y purinoceptors in hamster submandibular ganglion neurons.

1. The presence and profile of purinoceptors in neurons of the hamster submandibular ganglion (SMG) have been studied using the whole-cell configuration of the patch-clamp technique. 2. Extracellular application of adenosine 5'-triphosphate (ATP) reversibly inhibited voltage-dependent Ca(2+) channel (VDCC) currents (I(Ca)) via G(i/o)-protein in a voltage-dependent manner. 3. Extracellular application of uridine 5'-triphosphate (UTP), 2-methylthioATP (2-MeSATP), alpha,beta-methylene ATP (alpha,beta-MeATP) and adenosine 5'-diphosphate (ADP) also inhibited I(Ca). The rank order of potency was ATP=UTP>ADP>2-MeSATP=alpha,beta-MeATP. 4. The P2 purinoceptor antagonists, suramin and pyridoxal-5-phosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS), partially antagonized the ATP-induced inhibition of I(Ca), while coapplication of suramin and the P1 purinoceptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), virtually abolished I(Ca) inhibition. DPCPX alone partially antagonized I(Ca) inhibition. 5. Suramin antagonized the UTP-induced inhibition of I(Ca), while DPCPX had no effect. 6. Extracellular application of adenosine (ADO) also inhibited I(Ca) in a voltage-dependent manner via G(i/o)-protein activation. 7. Mainly N- and P/Q-type VDCCs were inhibited by both ATP and ADO via G(i/o)-protein betagamma subunits in seemingly convergence pathways.

G protein ?1?2 subunits purification and their interaction with adenylyl cyclase

A preliminary study on the interaction of G protein (guanine triphosphate binding protein) beta(1)gamma(2) subunits and their coupled components in cell signal transduction was conducted in vitro. The insect cell lines, Sf9 (Spodoptera frugiperda) and H5 (Trichoplusia ni) were used to express the recombinant protein Gbeta(1)gamma(2). The cell membrane containing Gbeta(1)gamma(2) was isolated through affinity chromatography column with Ni-NTAagarose by FPLC method, and the highly purified protein was obtained. The adenylyl cyclase 2 (AC(2)) activity assay showed that the purified Gbeta(1)gamma(2) could significantly stimulate AC(2) activity. The interaction of beta(1)gamma(2) subunits of G protein with the cytoplasmic tail of various mammalian adenylyl cyclases was monitored by BIAcore technology using NTA sensor chip, which relies on the phenomenon of surface plasmon resonance (SPR). The experiments showed the direct binding of Gbeta(1)gamma(2) to the cytoplasmic tail C2 domain of AC2. The specific binding domain of AC2 with Gbeta(1)gamma(2) was the same as AC2 activity domain which was stimulated by Gbeta(1)gamma(2).

Ghrelin and growth hormone (GH) secretagogues potentiate GH-releasing hormone (GHRH)-induced cyclic adenosine 3',5'-monophosphate production in cells expressing transfected GHRH and GH secretagogue receptors.

GHRH stimulates GH secretion from somatotroph cells of the anterior pituitary via a pathway that involves GHRH receptor activation of adenylyl cyclase and increased cAMP production. The actions of GHRH to release GH can be augmented by the synthetic GH secretagogues (GHS), which bind to a distinct G protein-coupled receptor to activate phospholipase C and increase production of the second messengers calcium and diacylglycerol. The stomach peptide ghrelin represents an endogenous ligand for the GHS receptor, which does not activate the cAMP signaling pathway. This study investigates the effects of GHS and ghrelin on GHRH-induced cAMP production in a homogenous population of cells expressing the cloned GHRH and GHS receptors. Each epitope-tagged receptor was shown to be appropriately expressed and to functionally couple to its respective second messenger pathway in this heterologous cell system. Although activation of the GHS receptor alone had no effect on cAMP production, coactivation of the GHS and GHRH receptors produced a cAMP response approximately twice that observed after activation of the GHRH receptor alone. This potentiated response is dose dependent with respect to both GHRH and GHS, is dependent on the expression of both receptors, and was observed with a variety of peptide and nonpeptide GHS compounds as well as with ghrelin-(1-5). Pharmacological inhibition of signaling molecules associated with GHS receptor activation, including G protein betagamma-subunits, phospholipase C, and protein kinase C, had no effect on GHS potentiation of GHRH-induced cAMP production. Importantly, the potentiation appears to be selective for the GHRH receptor. Treatment of cells with the pharmacological agent forskolin elevated cAMP levels, but these levels were not further increased by GHS receptor activation. Similarly, activation of two receptors homologous to the GHRH receptor, the vasoactive intestinal peptide and secretin receptors, increased cAMP levels, but these levels were not further increased by GHS receptor activation. Based on these findings, we speculate that direct interactions between the GHRH and GHS receptors may explain the observed effects on signal transduction.

Regulation of Angiotensin II-induced G Protein Signaling by Phosducin-like Protein

Phosducin-like protein (PhLP) is a broadly expressed member of the phosducin (Pd) family of G protein betagamma subunit (Gbetagamma)-binding proteins. Though PhLP has been shown to bind Gbetagamma in vitro, little is known about its physiological function. In the present study, the effect of PhLP on angiotensin II (Ang II) signaling was measured in Chinese hamster ovary cells expressing the type 1 Ang II receptor and various amounts of PhLP. Up to 3.6-fold overexpression of PhLP had no effect on Ang II-stimulated inositol trisphosphate (IP(3)) formation, whereas further increases caused an abrupt decrease in IP(3) production with half-maximal inhibition occurring at 6-fold PhLP overexpression. This threshold level for inhibition corresponds to the cellular concentration of cytosolic chaperonin complex, a recently described binding partner that preferentially binds PhLP over Gbetagamma. Results of pertussis toxin sensitivity, GTPgammaS binding, and immunoprecipitation experiments suggest that PhLP inhibits phospholipase Cbeta activation by dual mechanisms: (i) steric blockage of Gbetagamma activation of PLCbeta and (ii) interference with Gbetagamma-dependent cycling of G(q)alpha by the receptor. These results suggest that G protein signaling may be regulated through controlling the cellular concentration of free PhLP by inducing its expression or by regulating its binding to the chaperonin.

Modulation of a Brain Voltage-gated K+ Channel by Syntaxin 1A Requires the Physical Interaction of Gβγ with the Channel

Recently we suggested that direct interactions between voltage-gated K(+) channels and proteins of the exocytotic machinery, such as those observed between the Kv1.1/Kvbeta channel, syntaxin 1A, and SNAP-25 may be involved in neurotransmitter release. Furthermore, we demonstrated that the direct interaction with syntaxin 1A enhances the fast inactivation of Kv1.1/Kvbeta1.1 in oocytes. Here we show that G-protein betagamma subunits play a crucial role in the enhancement of inactivation by syntaxin 1A. The effect caused by overexpression of syntaxin 1A is eliminated in the presence of chelators of endogenous betagamma subunits in the whole cell and at the plasma membrane. Conversely, enhancement of inactivation caused by overexpression of beta(1)gamma(2) subunits is eliminated upon knock-down of endogenous syntaxin or its scavenging at the plasma membrane. We further show that the N terminus of Kv1.1 binds brain synaptosomal and recombinant syntaxin 1A and concomitantly binds beta(1)gamma(2); the binding of beta(1)gamma(2) enhances that of syntaxin 1A. Taken together, we suggest a mechanism whereby syntaxin and G protein betagamma subunits interact concomitantly with a Kv channel to regulate its inactivation.

Bi-directional Regulation of UV-induced Activation of p38 Kinase and c-Jun N-terminal Kinase by G Protein βγ-Subunits

Ultraviolet (UV) irradiation induces various cellular responses by activating many UV-responsive enzymes including mitogen-activated protein kinases (MAPKs). Various G protein-coupled receptor agonists also activate MAPKs, but it is not known whether or not G proteins also mediate the UV-induced activation of MAPKs. Therefore, this study was undertaken to determine whether the G protein betagamma-subunit (Gbetagamma) mediates the UV-induced activation of p38 and JNK. Gbetagamma overexpression in COS-1 cells amplified the UV-induced activation of p38 but reduced JNK activation. The overexpression of the C-terminal region of beta-adrenergic receptor kinase (betaARKct) decreased the UV-induced activation of p38 but increased JNK activation. Gbeta(1)gamma(2) expression increased MKK3/6 phosphorylation with a concomitant decrease in MKK4 phosphorylation, which contrasts with betaARKct expression. Gbeta(1)gamma(2) or betaARKct expression resulted in corresponding changes in the transcriptional activity of CHOP and c-Jun. Treatment with a p38 inhibitor, SB203580, or the expression of a kinase-inactive p38 increased the UV-induced JNK activation. Expression of the constitutively active MKK6 decreased the UV-induced JNK activation. In summary, although the endogenous Gbetagamma was found to mediate about half of the UV-induced activation of p38, it was found that exogenous Gbetagamma mediates the bi-directional regulation of UV-induced p38 and JNK activation, and that this bi-directional regulation results from the inhibition of JNK activation by the p38 activated via Gbetagamma in the COS-1 cells.

Regulatory interaction of phosducin-like protein with the cytosolic chaperonin complex.

Phosducin and phosducin-like protein (PhLP) bind G protein betagamma subunits and regulate their activity. This report describes a previously uncharacterized binding partner unique to PhLP that was discovered by coimmunoprecipitation coupled with mass spectrometric identification. Chaperonin containing tailless complex polypeptide 1 (CCT), a cytosolic chaperone responsible for the folding of many cellular proteins, binds PhLP with a stoichiometry of one PhLP per CCT complex. Unlike protein-folding substrates of CCT, which interact only in their nonnative conformations, PhLP binds in its native state. Native PhLP competes directly for binding of protein substrates of CCT and thereby inhibits CCT activity. Overexpression of PhLP inhibited the ability of CCT to fold newly synthesized beta-actin by 80%. These results suggest that the interaction between PhLP and CCT may be a means to regulate CCT-dependent protein folding or alternatively, to control the availability of PhLP to modulate G protein signaling.

Visualizing preference of G protein-coupled receptor kinase 3 for the process of kappa-opioid receptor sequestration.

G protein-coupled receptor kinases (GRKs) phosphorylate opioid receptors, which eventually results in receptor sequestration. With respect to kappa-opioid receptors, it is known that internalization occurs in a species-specific manner. That is, the agonist-occupied human kappa-receptors will sequester whereas murine receptors fail to do so. This investigation concentrates on the internalization of kappa-opioid receptors, employing laser scanning microscopy as a major technique to examine receptor internalization in living cells. For this reason, we fused green fluorescence protein to kappa-receptors, and DsRed-fluorescent protein to GRK2 and GRK3. All fusion proteins retained their biologic activities. Permanent cell lines (HEK 293, NG 108-15) were transfected to express either green fluorescent kappa-receptors or to coexpress the tagged receptor and a specific GRK-DsRed construct. The localization of fluorescent receptors and GRKs was monitored by confocal microscopy before and after opioid exposure of transfected cells. Activation of the murine kappa-receptors triggers rapid translocation of tagged GRKs toward the cell membrane, but receptor internalization was not observed. The agonist-occupied human kappa-receptor also causes translocation of GRK2- and GRK3-DsRed, which was followed by the formation of vesicles carrying the green fluorescent kappa-receptors. Moreover, the green fluorescent vesicles consistently harbour red fluorescent GRK2 and GRK3, respectively. The phenomenon of kappa-receptor internalization as well as cointernalization of GRKs is blocked by phosducin, indicating a critical role of G protein-betagamma subunits for kappa-receptor sequestration. Comparing the effect of over-expressed GRK2 and GRK3 on sequestration of kappa-receptors, we conclude that GRK3 more strongly induces kappa-receptor internalization than GRK2.

A glutamate residue at the C terminus regulates activity of inward rectifier K+ channels: implication for Andersen's syndrome.

G protein-coupled inward rectifiers (GIRKs) are activated directly by G protein betagamma subunits, whereas classical inward rectifiers (IRKs) are constitutively active. We found that a glutamate residue of GIRK2 (E315), located on a hydrophobic domain of the C terminus, is crucial for the channel activation. This glutamate (or aspartate) residue is conserved in all members of the Kir family. Substitution of alanine for the glutamate on GIRK1, GIRK2, and IRK2, expressed in HEK293 cells, greatly reduced the whole-cell currents. The whole-cell current of GIRK channels with a constitutively active gate, GIRK2(V188A), [Yi, B. A., Lin, Y. F., Jan, Y. N. & Jan, L. Y. (2001) Neuron 29, 657-667] was also reduced by the same glutamate mutation. Mean open time and conductance of single channels in GIRK2 and IRK2 were not affected by the mutation, indicating that the reduced whole-cell current resulted from a lowered probability of channel activation. The mutated GIRK and IRK showed normal trafficking to the cell membrane. The mutated GIRK2 retained the ability to interact with G protein betagamma subunits, and it showed almost the same inwardly rectifying property as the wild type. The mutated GIRK1 and GIRK2 retained ion selectivity to K(+) ions. This glutamate residue corresponds to one of the residues causing Andersen's syndrome [Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., et al. (2001) Cell 105, 511-519]. Our interpretation is that this region of the glutamate residue is crucial in relaying the activating message from the ligand sensor region to the gate.