Immunocytochemical and biochemical characterization of guanine nucleotide-binding regulatory proteins in mammalian spermatozoa

Polyclonal antisera directed against conserved and subtype-specific peptide sequences of the alpha-subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to characterize the nature of mammalian sperm G proteins and to determine whether their localization was consistent with their proposed roles in mediating ZP3-induced acrosomal exocytosis. Mouse and guinea pig sperm exhibit positive immunofluorescence in the acrosomal region using an antiserum directed against a peptide region common to all alpha-subunits of G proteins (G alpha). The immunofluorescence disappears after sperm have undergone the acrosome reaction, suggesting that the immunoreactive material is associated with the plasma membrane/outer acrosomal membrane region overlying the acrosome. The presence of G proteins in this region is confirmed by the presence of a Mr 41,000 substrate for pertussis toxin (PT)-catalyzed [32P]ADP-ribosylation in purified plasma membrane/outer acrosomal membrane hybrid vesicles obtained from acrosome-reacted guinea pig sperm. Immunoprecipitation and polyacrylamide gel electrophoresis of PT-catalyzed [32P]ADP-ribosylated protein(s) using anti-peptide antisera generated against sequences unique to Gi alpha 1, Gi alpha 2, and Gi alpha 3 confirm the existence of all three Gi subtypes in mouse sperm extracts. Indirect immunofluorescence using an antiserum directed against a peptide region present in Gz alpha, a PT-insensitive G protein, demonstrates positive immunoreactivity in the postacrosomal/lateral face region of the mouse sperm head. This immunoreactivity is retained during acrosomal exocytosis in response to solubilized ZP and then disappears subsequent to this exocytotic event. These data demonstrate that Gi protein alpha-subunits are present in the acrosomal region of mammalian sperm, consistent with their postulated role in regulating ZP3-mediated acrosomal exocytosis, and that PT-insensitive Gz alpha is found in a region of the sperm head distinct from that of the Gi alpha subunits.     

Molecular heterogeneity of the beta gamma-subunits of GTP-binding proteins in bovine brain membranes.

The guanine nucleotide-binding proteins (G proteins) are heterotrimers composed of alpha-, beta-, and gamma-subunits, and each of the constituent subunits has been reported to exhibit a molecular heterogeneity. The beta- and gamma-subunits form a functional unit that does not separate under physiological conditions and interact with various alpha-subunits that appear to mainly regulate specific effectors. We thus purified the beta gamma-complex of G proteins from bovine brain membranes and found that there were chromatographically multiple forms of beta gamma-subunits which could be reassociated with various alpha-subunits. The major findings observed with the purified proteins were summarized as follows. (a) The constituent beta gamma-subunits in the brain membrane G proteins appeared to be divided into two groups in their elution profiles from a hydrophobic column. (b) Each of the two groups contained at least five different components of beta gamma-subunits upon analyzing by a high-resolution, anion-exchange column. (c) Distribution of the heterogeneous beta gamma-subunits was not identical among various trimeric G proteins such as Gi, G0, and Gs. (d) The heterogeneous beta gamma-components were able to interact with a specific alpha-subunit resulting in the alpha beta gamma-trimer that served as the substrate of pertussis toxin-catalyzed ADP-ribosylation. (e) However, the apparent abilities of some beta gamma-subunits to support the toxin-induced modification were significantly different in a special comparison between the two beta gamma-groups that were eluted from the hydrophobic column. These results indicated that there were multiple forms of beta gamma-subunits associating with the specific alpha-subunit of a trimeric G protein and that some of those had different affinities for various alpha-subunits in terms of their tight associations. A possible role of the heterogeneity in beta gamma-subunits is also discussed in terms of G protein-mediated signal transductions.     

Molecular cloning and sequence determination of four different cDNA species coding for alpha-subunits of G proteins from Xenopus laevis oocytes

A cDNA library prepared from Xenopus laevis oocytes in lambda gt10 was screened with a mixture of three oligonucleotide probes designed to detect sequences found in different mammalian genes coding for alpha-subunits of G-proteins. In addition to a clone coding for a G alpha o-type subunit previously reported [(1989) FEBS Lett. 244, 188-192] four additional clones have been found coding for different G alpha protein subunits. By comparison with mammalian alpha-subunits, these oocyte cDNAs correspond to two closely related G alpha s-1a, to a G alpha i-1 and to a G alpha i-3 species. The derived amino acid sequences showed that both G alpha s species contain 379 residues, corresponding to the short species without the serine residue and with a calculated Mr of 42720. The G alpha i-1 gene encodes a 354 amino acid protein with an Mr of 39,000 and the G alpha i-3 encodes an incomplete open reading frame of 345 residues, lacking the first 9 amino acid residues at the NH2 terminus. All these G alpha-subunits showed high identity with their respective mammalian counterparts (75-80%), indicating a great degree of conservation through the evolution and the important cellular regulatory function that they play.     

Amino acid sequence determination of the novel forms of Go alpha purified from bovine brain membranes

Previously we have reported that there are at least four different forms of Go alpha in bovine brain membranes which can be distinguished by their elution profiles from Mono Q column and their immunological reactivities. The four alpha-subunits are referred to as alpha o1, alpha o2, alpha o3 and alpha o4 in their elution orders from the column. Partial amino acid sequences of the purified alpha o1 and alpha o2 were determined and compared with the predicted sequences of two classes of Go alpha cDNAs, termed Go alpha-1 and Go alpha-2. There were at least two unique fragments corresponding with the predicted amino acid sequence of the Go alpha-2 cDNA but different from that of the Go alpha-1 cDNA upon tryptic digestion of alpha o1- or alpha o2-subunit. The alpha o3- and alpha o4-subunits, but not alpha o1-and alpha o2-subunits, were recognized by an antibody raised against a unique amino acid sequence predicted from Go alpha-1 cDNA. These results suggest that alpha o1,2 subunits and alpha o3,4 subunits are encoded by Go alpha-2 cDNA and Go alpha-1 cDNA, respectively.     

Purification of GTP-binding proteins from bovine brain membranes. Identification of heterogeneity of the alpha-subunit of Go proteins

Using high-resolution Mono Q column chromatography, we purified 6 distinct peaks of GTP-binding proteins from bovine brain membranes. Five of them consisted of 3 polypeptides with alpha beta gamma-subunits and served as the substrate of islet-activating protein (IAP), pertussis toxin. The other one was purified as alpha-subunit alone and was also ADP-ribosylated by IAP in the presence of beta gamma-subunits. When each alpha-subunit was characterized by immunoblot analysis using various antibodies with defined specificity, the two of them were identified as Gi-1 and Gi-2, and other 4 appeared to be Go or Go-like G proteins. The alpha-subunits of immunologically Go-like proteins were apparently distinguishable from one another on elution profiles from the Mono Q column. Thus, there was a heterogeneity of the alpha-subunit of Go in the brain membranes.     

The G alpha q and G alpha 11 proteins couple the thyrotropin-releasing hormone receptor to phospholipase C in GH3 rat pituitary cells.

Thyrotropin-releasing hormone stimulates the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in GH3 cell membranes. The stimulation of the phosphoinositide phospholipase C (PI/PLC) activity can be blocked by incubation of GH3 membranes with polyclonal antibodies directed against a peptide derived from the C-terminal region of G alpha q and G alpha 11. Antibodies directed against the C-terminal region of other G alpha-subunits had no detectable effect. The inhibition was specific since addition of the peptide that was used to prepare the antibody completely reversed the inhibition. Further evidence for the coupling of the TRH receptor to G alpha q or G alpha 11 comes from a reconstitution experiment in which human embryonic kidney cells were transiently transfected with cDNAs corresponding to the TRH receptor, G alpha q or G alpha 11. The PIP2 hydrolysis detected with membranes from cells that over-expressed the TRH receptor alone was low, however, co-expression with the G alpha q or G alpha 11 subunits produced a synergistic stimulation of PI-PLC activity. In contrast, co-expression of these alpha-subunits with the M2 muscarinic acetylcholine receptor induced a weak stimulation of PIP2 hydrolysis. The results presented here suggest that the TRH-dependent stimulation of PI-PLC in GH3 cells is mediated through the G-protein alpha-subunits, G alpha q and/or G alpha 11.     

Evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts

Recent literature suggests that tetraspanin proteins (transmembrane 4 superfamily; TM4SF proteins) may associate with each other and with many other transmembrane proteins to form large complexes that sometimes may be found in lipid rafts. Here we show that prototype complexes of CD9 or CD81 (TM4SF proteins) with alpha(3)beta(1) (an integrin) and complexes of CD63 (a TM4SF protein) with phosphatidylinositol 4-kinase (PtdIns 4-K) may indeed localize within lipid raft-like microdomains, as seen by three different criteria. First, these complexes localize to low density light membrane fractions in sucrose gradients. Second, CD9 and alpha(3) integrin colocalized with ganglioside GM1 as seen by double staining of fixed cells. Third, CD9-alpha3beta1 and CD81-alpha3beta1 complexes were shifted to a higher density upon cholesterol depletion from intact cells or cell lysate. However, CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K complex formation itself was not dependent on localization into raftlike lipid microdomains. These complexes did not require cholesterol for stabilization, were maintained within well solubilized dense fractions from sucrose gradients, were stable at 37 degrees C, and were small enough to be included within CL6B gel filtration columns. In summary, prototype TM4SF protein complexes (CD9-alpha3beta1, CD81-alpha3beta1, and CD63-PtdIns 4-K) can be solubilized as discrete units, independent of lipid microdomains, although they do associate with microdomains resembling lipid rafts.     

Decreased levels of guanyl nucleotide-binding regulatory protein alpha-subunits in Y1 adrenocortical tumor cell mutants resistant to forskolin.

Forskolin-resistant mutants arise from Y1 mouse adrenocortical tumor cells with a frequency indicative of a mutational event at a single genetic locus and exhibit adenylyl cyclases that are resistant to activation by forskolin, corticotropin, and guanyl-5'-yl-imidodiphosphate. This study examined the levels of guanyl nucleotide-binding regulatory protein subunits (G) in plasma membranes from the forskolin-resistant mutants by Western blot immunoanalysis. In plasma membranes prepared from parental Y1 cells and from four forskolin-resistant mutants, 10r-2, 10r-3, 10r-6, and 10r-9, the levels of the alpha-subunits of Gs and Gi-2 were reduced by 70-80% relative to the levels in parental Y1 cells. The levels of the beta 36-subunit were much less affected, and the levels of the alpha i-3 and beta 35-subunits varied independently of the forskolin-resistant phenotype. As determined by slot blot hybridization analyses, the levels of Gs alpha and Gi alpha RNA in the forskolin-resistant mutants were equivalent to those in the Y1 parent. Therefore, the decreased levels of Gs alpha and Gi alpha-2 subunits observed in the forskolin-resistant mutants did not result from decreased expression of the genes encoding these proteins. Our observations suggest that the forskolin-resistant phenotype of Y1 mutants resulted from single mutations that affected the processing of specific G alpha subunits or their incorporation into the plasma membrane.     

A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go

A1 adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) were purified from bovine cerebral cortex by affinity chromatography (Munshi, R., and Linden, J. (1989) J. Biol. Chem. 264, 14853-14859). In this study we have identified the pertussis toxin-sensitive G protein subunits that co-purify with A1 adenosine receptors by immunoblotting with specific antipeptide antisera. Gi alpha 1, Gi alpha 2, Go alpha, G beta 35, and G beta 36 were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate [( 35S]GTP gamma S) binding sites, Gi alpha 1 and Go alpha each accounted for greater than 37% whereas Gi alpha 2 comprised less than 13%. G beta 35 was found in excess over G beta 36. Low molecular mass (21-25 kDa) GTP-binding proteins were not detected. We also examined the characteristics of purified receptors and various purified bovine brain G proteins reconstituted into phospholipid vesicles. All three alpha-subunits restored GTP gamma S-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of Gi2 greater than Go greater than or equal to Gi1 (ED50 values of G proteins measured as fold excess over the receptor concentration were 4.7 +/- 1.2, 24 +/- 5, and 34 +/- 7, respectively). Furthermore, receptors occupied with the agonist R-phenylisopropyladenosine catalytically increased the rate of binding of [35S]GTP gamma S to reconstituted G proteins by 6.5-8.5-fold. These results suggest that A1 adenosine receptors couple indiscriminately to pertussis toxin-sensitive G proteins.     

Functional interactions of recombinant alpha 2 adrenergic receptor subtypes and G proteins in reconstituted phospholipid vesicles.

The functional interaction of the recombinant alpha 2 adrenergic receptor subtypes, alpha 2-C10 (the human platelet alpha 2 receptor, equivalent to the alpha 2 A subtype) and alpha 2-C4 (an alpha 2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1, Gi2, and Gi3, and G0. The cloned alpha subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the alpha subunits with beta gamma subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both alpha 2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the alpha 2-C10 adrenergic receptor subtype showed the following preference: Gi3 greater than Gi1 greater than or equal to Gi2 greater than G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the alpha 2-C4 receptor subtype was the same as that observed for the alpha 2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the alpha 2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that alpha 2-C10 is coupled more efficiently to G-protein activation than is alpha 2-C4.     

Interaction between HIV-1 NEF and G(o) proteins in transfected COS-7 cells

Nef protein of HIV/SIV lentiviruses affects G-protein-mediated signaling, and physically associates to Lck, a myristoylated and palmitoylated Src-like tyrosine kinase. To assess whether Nef interacts with alpha-subunits of heterotrimeric G proteins (Galpha), carrying the same lipidation motif as Lck, we transiently expressed Nef and G(o)alpha (wild-type or nonpalmitoylated C3S mutant), individually or in combination, in transfected COS-7 cells. Recombinant Nef was mostly recovered in particulate fractions, and a Nef-Green Fluorescent Protein chimera was localized at the plasmalemma by in vivo fluorescence imaging. Moreover, Nef and C3S were entirely solubilized by cold Triton X-100, and excluded from low buoyant density sucrose gradient fractions, containing caveolin-1, whereas wild-type G(o)alpha was partially resistant to Triton extraction, and colocalized with caveolin-1. After coexpression, Nef recruited soluble C3S to membranes, and the two proteins were coimmunoprecipitated by G(o)alpha and Nef antisera. We conclude that Nef interacts with nonpalmitoylated G(o)alpha, presumably outside caveolin-rich microdomains of the plasma membrane.     

Identification in bovine liver plasma membranes of a Gq-activatable phosphoinositide phospholipase C.

Phosphoinositide phospholipase C (PLC) activity extracted from bovine liver plasma membranes with sodium cholate was stimulated by GTP gamma S-activated G alpha q/G alpha 11, whereas the enzyme from liver cytosol was not. The membrane-associated PLC was subjected to chromatography on heparin-Sepharose, Q Sepharose, and S300HR, enabling the isolation of the G-protein stimulated activity and its resolution from PLC-gamma and PLC-delta. Following gel filtration, two proteins of 150 and 140 kDa were found to correspond to the activatable enzyme. These proteins were identified immunologically as members of the PLC-beta family and were completely resolved by chromatography on TSK Phenyl 5PW. The 150-kDa enzyme was markedly responsive to GTP gamma S-activated alpha-subunits of G alpha q/G alpha 11 or to purified Gq/G11 in the presence of GTP gamma S. The response of this PLC was of much greater magnitude than that of the 140-kDa enzyme. The partially purified 150-kDa enzyme showed specificity for PtdIns(4,5)P2 and PtdIns4P as compared to PtdIns and had an absolute dependence upon Ca2+. These characteristics were similar to those of the brain PLC-beta 1. The immunological and biochemical properties of the 150-kDa membrane-associated enzyme are consistent with its being the PLC-beta isozyme that is involved in receptor-G-protein-mediated generation of inositol 1,4,5-triphosphate in liver.     

Insulin-dependent phosphorylation of GTP-binding proteins in phospholipid vesicles

The involvement of GTP-binding proteins (G proteins) in insulin action has been investigated in an in vitro system. Insulin receptors that have been purified by wheat germ lectin chromatography and either tyrosine-agarose chromatography, sucrose density centrifugation, or insulin-Sepharose chromatography have been co-inserted into phospholipid vesicles with different purified G proteins. The results of these studies indicate that a specific insulin-promoted phosphorylation of two G proteins, Go and Gi, can occur in these phospholipid vesicles. Bovine retinal transducin is a poor substitute for Go and Gi, being only weakly phosphorylated by the insulin receptor, and bovine brain Gs is not a substrate. The phosphorylation of Gi and Go occurs primarily on the alpha-subunits. Under optimal conditions, about one alpha o- or alpha i-subunit is phosphorylated on a tyrosine residue for every two beta-subunits of the insulin receptor, suggesting a 1:1 interaction between these G proteins and the heterotetrameric (alpha 2 beta 2) insulin receptor molecular. The inactive (GDP-bound) form of the alpha-subunits appears to be the preferred substrate, with the phosphorylation being significantly reduced in alpha o and alpha i upon the binding of guanosine 5'-O-thiotriphosphate (GTP gamma S) and completely eliminated in the pure alpha-GTP gamma S complex of transducin. The Gi and Go proteins also cause an enhancement of the insulin-stimulated receptor autophosphorylation. This enhancement is a reflection of an increased incorporation of the insulin receptor into lipid vesicles which is induced by these G proteins. Taken together these results provide evidence for the interactions of G proteins with the insulin receptor in a lipid milieu.     

Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli

Complementary DNAs for the G protein alpha subunits Gi alpha 1, Gi alpha 2, Gi alpha 3, and Go alpha were expressed in Escherichia coli, and the four proteins were purified to homogeneity. The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with beta gamma subunits. The rates of dissociation of GDP from Gi alpha 1 and Gi alpha 3 (0.03 min-1) are an order of magnitude slower than that from rGo alpha; release of GDP from Gi alpha 2 is also relatively slow (0.07 min-1). However, the values of kcat for the hydrolysis of GTP by rGo alpha and the three rGi alpha proteins are approximately the same, about 2 min-1 at 20 degrees C. The recombinant proteins restore inhibition of Ca2+ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system. The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin, while Gi alpha 2 was inactive with neuropeptide Y. Despite these results, high concentrations of activated Gi alpha proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or Gs alpha, the G protein that activates adenylyl cyclase. These results are consistent with the hypothesis that G protein beta gamma subunits are primarily responsible for inhibition of adenylyl cyclase activity.     

Molecular basis for HbH disease in Italy: geographical distribution of deletional and nondeletional alpha-thalassemia haplotypes.

We have investigated the molecular basis for HbH disease in 16 patients from Sardinia, and central and southern Italy. We have shown that HbH disease is produced by the interaction of at least 10 different deletional or nondeletional alpha-thalassemia haplotypes, some of which have been already described in the Mediterranean area (--Med,-(alpha)20.5,-alpha 3.7 type I,-alpha 3.7 type II, alpha 2 NcoI alpha 1, alpha 2 HphI alpha 1). Among the new mutations found in the course of our study, there is a complete deletion of the zeta-alpha cluster and three nondeletional determinants (alpha alpha T), affecting to various extents alpha-globin gene expression. The different alpha-thalassemia haplotypes are not evenly distributed throughout the country. Two alpha 0 determinants [-(alpha)20.5 and the complete deletion of the zeta-alpha cluster] and four alpha + determinants (-alpha 3.7 type II, three nondeletional alpha alpha T mutations) are found exclusively in southern Italy.     

Lack of effect of incretin hormones on insulin release from pancreatic islets in the bile duct-ligated rats

Hyperglycemia associated with obstructive jaundice seriously affects the prognosis of patients with hepatobiliary diseases. We investigated secretory properties of isolated islets from bile duct-ligated (BDL) rats. Pancreatic islets from BDL rats lost their secretory responses to glucagon-like peptide-1 (GLP-1), although their responses to glucose were normal. Loss of potentiation of insulin release was also observed in glucagon and glucose-dependent insulinotropic peptide (GIP), whereas modulation of the release by forskolin, dibutyryl cAMP, or epinephrine remained unaffected. cAMP production by BDL islets was not increased by these insulinotropic hormones. Serum levels of glucagon, but not GIP, were increased in BDL rats. GLP-1 levels were also elevated, although they did not reach statistical significance. Immunoblotting of trimeric G protein subunits demonstrated that G(s)alpha L and G(s)alpha S, but not G(i)alpha 1/2 and G(i)alpha 3/o alpha, were less expressed in BDL islets. Therefore, unresponsiveness of the beta-cell to cAMP-raising hormones is involved in glucose intolerance under cholestasis. It results from diminished expression of alpha-subunits of the relevant G protein, G(s), and desensitization of receptors of these hormones.