Identification and localization of G protein subunits in human spermatozoa.
Antibodies to alpha and beta subunits of guanine nucleotide regulatory proteins (G proteins) were used to identify which G proteins are present in mature human spermatozoa and to determine their subcellular localization. Immunoblots of membranes from spermatozoa demonstrate the presence of Galphai2, Galphai3, Galphaq/11 and Gbeta35 and the absence of Galphai1, Galpha0, Galphas, Galpha12, Galpha13, Galpha16, Galpha and Gbeta36. Indirect immunofluorescence demonstrates the presence of Galphaq/11 in the acrosome, with the highest proportion in the equatorial segment. Galphai2 is present in the acrosome, midpiece and tailpiece and Galphai3 in the postnuclear cap, midpiece and tailpiece. The Gbeta35 subunit is found mostly in the midpiece, with marginal labelling of the head, tailpiece and the equatorial segment of the acrosome. The distinct pattern of distribution of G proteins suggests that they may couple to receptors or effectors which also have discrete regions of localization in spermatozoa. These highly localized signal transduction pathways may regulate discrete functions, such as activation of the acrosome reaction, fusion with the oocyte and motility. (+info)
Identification of Galpha13 as one of the G-proteins that couple to human platelet thromboxane A2 receptors.
Previous studies have shown that ligand or immunoaffinity chromatography can be used to purify the human platelet thromboxane A2 (TXA2) receptor-Galphaq complex. The same principle of co-elution was used to identify another G-protein associated with platelet TXA2 receptors. It was found that in addition to Galphaq, purification of TXA2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the co-purification of a member of the G12 family. Using an antipeptide antibody specific for the human G13 alpha-subunit, this G-protein was identified as Galpha13. In separate experiments, it was found that the TXA2 receptor agonist U46619 stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) incorporation into G13 alpha-subunit. Further evidence for functional coupling of G13 to TXA2 receptors was provided in studies where solubilized platelet membranes were subjected to immunoaffinity chromatography using an antibody raised against native TXA2 receptor protein. It was found that U46619 induced a significant decrease in Galphaq and Galpha13 association with the receptor protein. These results indicate that both Galphaq and Galpha13 are functionally coupled to TXA2 receptors and dissociate upon agonist activation. Furthermore, this agonist effect was specifically blocked by pretreatment with the TXA2 receptor antagonist, BM13.505. Taken collectively, these data provide direct evidence that endogenous Galpha13 is a TXA2 receptor-coupled G-protein, as: 1) its alpha-subunit can be co-purified with the receptor protein using both ligand and immunoaffinity chromatography, 2) TXA2 receptor activation stimulates GTPgammaS binding to Galpha13, and 3) Galpha13 affinity for the TXA2 receptor can be modulated by agonist-receptor activation. (+info)
Stimulation of cAMP synthesis by Gi-coupled receptors upon ablation of distinct Galphai protein expression. Gi subtype specificity of the 5-HT1A receptor.
The three Galphai subunits were independently depleted from rat pituitary GH4C1 cells by stable transfection of each Galphai antisense rat cDNA construct. Depletion of any Galphai subunit eliminated receptor-induced inhibition of basal cAMP production, indicating that all Galphai subunits are required for this response. By contrast, receptor-mediated inhibition of vasoactive intestinal peptide (VIP)-stimulated cAMP production was blocked by selective depletions for responses induced by the transfected serotonin 1A (5-HT1A) (Galphai2 or Galphai3) or endogenous muscarinic-M4 (Galphai1 or Galphai2) receptors. Strikingly, receptor activation in Galphai1-depleted clones (for the 5-HT1A receptor) or Galphai3-depleted clones (for the muscarinic receptor) induced a pertussis toxin-sensitive increase in basal cAMP production, whereas the inhibitory action on VIP-stimulated cAMP synthesis remained. Finally, in Galphai2-depleted clones, activation of 5-HT1A receptors increased VIP-stimulated cAMP synthesis. Thus, 5-HT1A and muscarinic M4 receptor may couple dominantly to Galphai1 and Galphai3, respectively, to inhibit cAMP production. Upon removal of these Galphai subunits to reduce inhibitory coupling, stimulatory receptor coupling is revealed that may involve Gbetagamma-induced activation of adenylyl cyclase II, a Gi-stimulated cyclase that is predominantly expressed in GH4C1 cells. Thus Gi-coupled receptor activation involves integration of both inhibitory and stimulatory outputs that can be modulated by specific changes in alphai subunit expression level. (+info)
Differential involvement of Galpha12 and Galpha13 in receptor-mediated stress fiber formation.
The ubiquitously expressed heterotrimeric guanine nucleotide-binding proteins (G-proteins) G12 and G13 have been shown to activate the small GTPase Rho. Rho stimulation leads to a rapid remodeling of the actin cytoskeleton and subsequent stress fiber formation. We investigated the involvement of G12 or G13 in stress fiber formation induced through a variety of Gq/G11-coupled receptors. Using fibroblast cell lines derived from wild-type and Galphaq/Galpha11-deficient mice, we show that agonist-dependent activation of the endogenous receptors for thrombin or lysophosphatidic acid and of the heterologously expressed bradykinin B2, vasopressin V1A, endothelin ETA, and serotonin 5-HT2C receptors induced stress fiber formation in either the presence or absence of Galphaq/Galpha11. Stress fiber assembly induced through the muscarinic M1 and the metabotropic glutamate subtype 1alpha receptors was dependent on Gq/G11 proteins. The activation of the Gq/G11-coupled endothelin ETB and angiotensin AT1A receptors failed to induce stress fiber formation. Lysophosphatidic acid, B2, and 5-HT2C receptor-mediated stress fiber formation was dependent on Galpha13 and involved epidermal growth factor (EGF) receptors, whereas thrombin, ETA, and V1A receptors induced stress fiber accumulation via Galpha12 in an EGF receptor-independent manner. Our data demonstrate that many Gq/G11-coupled receptors induce stress fiber assembly in the absence of Galphaq and Galpha11 and that this involves either a Galpha12 or a Galpha13/EGF receptor-mediated pathway. (+info)
Modulation of rat cardiac sodium channel by the stimulatory G protein alpha subunit.
1. Modulation of cardiac sodium currents (INa) by the G protein stimulatory alpha subunit (Gsalpha) was studied using patch-clamp techniques on freshly dissociated rat ventricular myocytes. 2. Whole-cell recordings showed that stimulation of beta-adrenergic receptors with 10 microM isoprenaline (isoproterenol, ISO) enhanced INa by 68.4 +/- 9.6 % (mean +/- s.e.m.; n = 7, P < 0.05 vs. baseline). With the addition of 22 microgram ml-1 protein kinase A inhibitor (PKI) to the pipette solution, 10 microM ISO enhanced INa by 30.5 +/- 7.0 % (n = 7, P < 0.05 vs. baseline). With the pipette solution containing both PKI and 20 microgram ml-1 anti-Gsalpha IgG or 20 microgram ml-1 anti-Gsalpha IgG alone, 10 microM ISO produced no change in INa. 3. The effect of Gsalpha on INa was not due to changes in the steady-state activation or inactivation curves, the time course of current decay, the development of inactivation, or the recovery from inactivation. 4. Whole-cell INa was increased by 45.2 +/- 5.3% (n = 13, P < 0.05 vs. control) with pipette solution containing 1 microM Gsalpha27-42 peptide (amino acids 27-42 of rat brain Gsalpha) without altering the properties of Na+ channel kinetics. Furthermore, application of 1 nM Gsalpha27-42 to Na+ channels in inside-out macropatches increased the ensemble-averaged INa by 32.5 +/- 6.8 % (n = 8, P < 0.05 vs. baseline). The increase in INa was reversible upon Gsalpha27-42 peptide washout. Single channel experiments showed that the Gsalpha27-42 peptide did not alter the Na+ single channel current amplitude, the mean open time or the mean closed time, but increased the number of functional channels (N) in the patch. 5. Application of selected short amino acid segments (Gsalpha27-36, Gsalpha33-42 and Gsalpha30-39) of the 16 amino acid Gsalpha peptide (Gsalpha27-42 peptide) showed that only the C-terminal segment of this peptide (Gsalpha33-42) significantly increased INa in a dose-dependent fashion. These results show that cardiac INa is regulated by Gsalpha via a mechanism independent of PKA that results in an increase in the number of functional Na+ channels. In addition, a 10 residue domain (amino acids 33-42) near the N-terminus of Gsalpha is important in modulating cardiac Na+ channels. (+info)
The Src family tyrosine kinase is involved in Rho-dependent activation of c-Jun N-terminal kinase by Galpha12.
Gt12, a member of alpha subunit of heterotrimeric G protein G12 subfamily, has been shown to stimulate c-Jun N-terminal kinase (JNK) activity through the low molecular weight GTP-binding proteins Ras, Rac, and Cdc42. In this study using the transient expression of a constitutively activated mutant of Galpha12 (Galpha12Q229L) in human embryonic kidney (HEK) 293 cells, we found that Rho and Src family kinase are also involved in the Galpha12-induced activation of JNK. The activation of JNK by Galpha12Q229L was inhibited by dominant-negative RhoA(T19N), and botulinum C3 exoenzyme which specifically inactivates Rho. In addition, the expression of activated RhoA(G14V) elevated JNK activity in HEK 293 cells. The Galpha12Q229L-stimulated activation of JNK was blocked by a specific inhibitor of protein tyrosine kinases (PP2), and C-terminal Src kinase (Csk). Moreover, we observed that Galpha12Q229L stimulated Src family kinase activity and v-Src induced JNK activation. Interestingly, the v-Src-induced activation of JNK was inhibited by dominant-negative RhoA(T19N). In contrast, Csk did not inhibit the JNK activation by activated RhoA(G14V). These results suggest that Rho and Src family kinase are required for the Galpha12-induced JNK activation, and that Src family kinase acts upstream of Rho activation in the JNK pathway. (+info)
Simultaneous Cre catalyzed recombination of two alleles to restore neomycin sensitivity and facilitate homozygous mutations.
Cells homozygous for neo-expressing mutations can be derived by culturing heterozygotes with elevated G418. We demonstrate that this strategy is significantly less efficient if hyg is substituted for neo. Therefore, to introduce additional mutations Cre recombinase was used to remove floxed neo from both alleles of homozygotes at two different loci. The rate-determining step in Cre excision appeared independent of substrate copy number. Incorporating cytosine deaminase and Herpes simplex virus thymidine kinase allowed negative selection for both targeting and Cre excision. The resulting G418-sensitive homozygous mutants should allow mutagenesis at additional loci and avoid untoward effects of retained selection markers. (+info)
A rho exchange factor mediates thrombin and Galpha(12)-induced cytoskeletal responses.
Thrombin induces astrocytoma cell rounding through a Rho-dependent pathway (Majumdar, M., Seasholtz, T. M., Goldstein, D., de Lanerolle, P., and Brown, J. H. (1998) J. Biol. Chem. 273, 10099-10106). The involvement of the G(12) family of G proteins and the role of specific Rho exchange factors in transducing signals from the thrombin receptor to Rho-dependent cytoskeletal responses was examined. Microinjection of cDNAs for activated Galpha(12) or Galpha(13) induced cell rounding, and antibodies to Galpha(12) or Galpha(13) blocked the response to thrombin. In contrast, activation or inhibition of Galpha(q) function had relatively little effect. The cytoskeletal response to Galpha(12) was inhibited by microinjection of C3 exoenzyme, indicating Rho dependence. Two Rho-specific guanine nucleotide exchange factors (GEFs), oncogenic lbc and p115, increased the percentage of rounded cells 4-5-fold, and this was inhibited by C3. Mutant GEFs lacking the Dbl homology (DH) domain required for exchange factor activity failed to induce cell rounding. However, the DH mutants of lbc and p115 were efficacious inhibitors of rounding induced by thrombin or Galpha(12). The effects of lbc were dependent on an intact pleckstrin homology domain, which may be required for appropriate targeting of the Rho-GEF. These findings identify the Galpha(12) protein family as transducers of thrombin signaling to the cytoskeleton and provide the first evidence that a Rho-GEF transduces signals between G protein-coupled receptors and Rho-mediated cytoskeletal responses. (+info)