In vitro synthesis of G protein beta gamma dimers

The guanine nucleotide-binding proteins (G proteins), which play a central role in coupling membrane-bound receptors to intracellular effectors, are heterotrimers composed of alpha, beta, and gamma subunits. The beta and gamma subunits form a functional monomer that does not appear to separate under physiological conditions. This has made it difficult to differentiate the individual roles of beta and gamma subunits in signal transduction. To characterize the individual subunits, the 36-kDa beta subunit (beta 1), brain gamma (gamma 2), and transducin gamma (gamma t) were translated in vitro in a rabbit reticulocyte lysate system. Hydrodynamic studies and tryptic proteolysis were used to compare the physical properties of the in vitro translation products with those of beta gamma dimers purified from bovine brain. The hydrodynamic studies indicate that, without gamma subunits, the beta subunits are not stable but tend to aggregate into high molecular weight complexes. When beta and gamma subunits were co-translated, stable beta gamma dimers formed that bound alpha 0 in a guanine nucleotide-dependent manner. The beta gamma dimers were less hydrophobic than those purified from bovine brain. This may reflect a lack of post-translational modification in the reticulocyte lysate or other differences between the in vitro translation products and the purified beta gamma. When beta and gamma were translated separately and then mixed, beta gamma dimers also formed. Analysis of in vitro translated beta gamma subunits will provide ways to assess the function of these subunits and to determine the structural requirements for beta gamma formation.     

Modification of nuclear lamin proteins by a mevalonic acid derivative occurs in reticulocyte lysates and requires the cysteine residue of the C-terminal CXXM motif.

The C-terminus of nuclear lamins (CXXM) resembles a C-terminal motif (the CAAX box) of fungal mating factors and ras-related proteins. The CAAX box is subject to different types of post-translational modifications, including proteolytic processing, isoprenylation and carboxyl methylation. By peptide mapping we show that both chicken lamins A and B2 are processed proteolytically in vivo. However, whereas the entire CXXM motif is cleaved from lamin A, at most three C-terminal amino acids are removed from lamin B2. Following translation of cDNA-derived RNAs in reticulocyte lysates, lamin proteins specifically incorporate a derivative of [14C]mevalonic acid (MV), i.e. the precursor of a putative isoprenoid modification. Remarkably, no MV is incorporated into lamin B2 translated from a mutant cDNA encoding alanine instead of cysteine in the C-terminal CXXM motif. These results implicate this particular cysteine residue as the target for modification of lamin proteins by an isoprenoid MV derivative, and they indicate that isoprenylation is amenable to studies in cell-free systems. Moreover, our observations suggest that C-terminal processing of newly synthesized nuclear lamins is a multi-step process highly reminiscent of the pathway elaborated recently for ras-related proteins.     

Carboxyl-terminal isoprenylation of ras-related GTP-binding proteins encoded by rac1, rac2, and ralA

Membrane localization of p21ras is dependent upon its posttranslational modification by a 15-carbon farnesyl group. The isoprenoid is linked to a cysteine located within a conserved carboxyl-terminal sequence termed the "CAAX" box (where C is cysteine, A is an aliphatic amino acid, and X is any amino acid). We now show that three GTP-binding proteins encoded by the recently identified rac1, rac2, and ralA genes also undergo isoprenoid modification. cDNAs coding for each protein were transcribed in vitro, and the RNAs were translated in reticulocyte lysates. Incorporation of isoprenoid precursors, [3H]mevalonate or [3H]farnesyl pyrophosphate, indicated that the translation products were modified by isoprenyl groups. A protein recognized by an antibody to rac1 also comigrated with a protein metabolically labeled by a product of [3H] mevalonate in cultured cells. Gel permeation chromatography of radiolabeled hydrocarbons released from the rac1, rac2, and ralA proteins by reaction with Raney nickel catalyst indicated that unlike p21Hras, which was modified by a 15-carbon moiety, the rac and ralA translation products were modified by 20-carbon isoprenyl groups. Site-directed mutagenesis established that the isoprenylated cysteines in the rac1, rac2, and ralA proteins were located in the fourth position from the carboxyl terminus. The three-amino acid extension distal to the cysteine was required for this modification. The isoprenylation of rac1 (CSLL), ralA (CCIL), and the site-directed mutants rac1 (CRLL) and ralA (CSIL), demonstrates that the amino acid adjacent to the cysteine need not be aliphatic. Therefore, proteins with carboxyl-terminal CXXX sequences that depart from the CAAX motif should be considered as potential targets for isoprenoid modification.     

Identification and isolation of common and tissue-specific geranylgeranylated gamma subunits of guanine-nucleotide-binding regulatory proteins in various tissues.

Heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) have been classified into several subtypes on the basis of the properties of their alpha subunits, though a notable multiplicity of gamma subunits has also been demonstrated. To investigate whether each subtype of alpha subunit is associated with a particular gamma subunit, various oligomeric G proteins, purified from bovine tissues, were subjected to gel electrophoresis in a Tricine buffer system. All G proteins examined were shown to have more than two kinds of gamma subunit. Of the brain G proteins, GoA, GoB, and Gi1 contain the same set of three gamma subunits, but Gi2 contains only two of these subunits. Lung Gi1 and Gi2 and spleen Gi2 and Gi3 had similar sets of two gamma subunits, one of which was distinct from the gamma subunits of brain G proteins. These observations indicate that each subtype of alpha subunit is associated with a variety of beta gamma subunits, and that the combinations differ among cells. For analyses of the structural diversity of the gamma subunits, beta gamma subunits were purified from the total G proteins of each tissue and subjected to reverse-phase HPLC under denaturing conditions, where none of the beta subunits were eluted from the column. Three distinct gamma subunits were isolated in this way from brain beta gamma subunits. In contrast, lung and spleen beta gamma subunits contained at least five gamma subunits, the elution positions and electrophoretic mobilities of which were indistinguishable between the two tissues. Among several gamma subunits, two subspecies appeared to be common to the three tissues. In fact, in each case, the partial amino acid sequence of the most abundant gamma subunit in each tissue was identical, and the sequences coincided exactly with that of 'gamma 6' [Robishaw, J. D., Kalman, V. K., Moomaw, C. R. & Slaughter, C. A. (1989) J. Biol. Chem. 264, 15758-15761]. Fast-atom-bombardment mass spectrometry analysis indicated that this abundant gamma subunit in lung and spleen was geranylgeranylated and carboxymethylated at the C-terminus, as was 'gamma 6' from brain. In addition to abundant gamma subunits, other tissue-specific gamma subunits were also shown to be geranylgeranylated by gas-chromatography-coupled mass spectrometry analysis of Raney nickel-treated gamma subunits. These results suggest that most gamma subunits associated with many different subtypes of alpha subunit are geranylgeranylated in a variety of tissues, with the single exception being the retina where the G protein transducin has a farnesylated gamma subunit.     

Existence of two gamma subunits of the G proteins in brain

Although amino acid sequences have been determined for the alpha and beta subunits of Gs, Gi, and Go, sequences have not been reported for the gamma subunits of these G proteins. In the present paper, we determined the sequences of peptides prepared by partial proteolysis of two different forms of the gamma subunit of Gs, Gi, and Go from bovine brain. Using oligonucleotide probes based on the sequences of two of these peptides, a cDNA clone was isolated from a bovine adrenal cDNA library. This clone contained a 0.9-kilobase cDNA insert that included an open reading frame of 213 bases encoding a 71-amino acid polypeptide with an estimated Mr of 7850. The amino acid sequence predicted for the adrenal cDNA clone was identical to that determined for one form of the gamma subunit from brain. In addition, an antibody to a peptide based on the predicted amino acid sequence of this cDNA clone reacted specifically with one of the brain gamma subunits, indicating the adrenal cDNA clone encodes a gamma subunit present in both adrenal gland and brain. Also, evidence is presented, demonstrating the existence of a second, structurally distinct, form of the gamma subunit of Gs, Gi, and Go in brain.     

Mutagenesis of the amino terminus of the alpha subunit of the G protein Go. In vitro characterization of alpha o beta gamma interactions.

Heterotrimeric guanine nucleotide-binding proteins are composed of alpha and beta gamma subunits and couple a variety of cell-surface receptors to intracellular enzymes or ion channels. The heterotrimer dissociates into alpha and beta gamma subunits when the alpha subunit is activated by guanine nucleoside triphosphates. Several lines of evidence show that the amino terminus of the alpha subunit is important for the interaction with the beta gamma subunit (Neer, E. J., Pulsifer, L., and Wolf, L. G. (1988) J. Biol. Chem. 263, 8996-9000; Fung, B. K.-K., and Nash, C. R. (1983) J. Biol. Chem. 258, 10503-10510). We have mutagenized the amino terminus of alpha o to dissect the relative contributions of amino-terminal myristoylation and specific amino acid sequences to subunit interaction. Wild-type and mutant alpha o cDNAs were translated in vitro in a rabbit reticulocyte lysate. All proteins were able to bind guanosine 5'-(gamma-thio)triphosphate and to achieve the necessary conformation for protection from tryptic digestion. Two assays of alpha o beta gamma interactions were used: sucrose density gradients to look for stable heterotrimer formation and ADP-ribosylation by pertussis toxin to detect weak or transient alpha o beta gamma interactions. Our results indicate that myristoylation is essential for stable heterotrimer formation, but that nonmyristoylated proteins are also capable of interacting with the beta gamma subunit. Amino acids 7-10 have an important role in alpha o beta gamma interactions whether alpha o is myristoylated or not. Deletion of this region diminishes the ability of alpha o to interact with the beta gamma subunit, but substitutions at this position indicate that other amino acids can be tolerated without affecting subunit interaction.     

Molecular cloning of a new human G protein. Evidence for two Gi alpha-like protein families

The amino acid sequence of a novel G protein alpha subunit (Gx alpha) has been deduced from the nucleotide sequence of a human cDNA clone isolated from a differentiated HL-60 cDNA library. The cDNA encodes a polypeptide of 354 amino acids (Mr 40,519) which is closely related to Gi alpha proteins. The amino acid sequence homology between Gx alpha and human myeloid Gi alpha is 86% with 15 nonconservative substitutions. Gx alpha also shares 86% homology with both rat brain and mouse macrophage Gi alpha but is more homologous (94%) to bovine brain Gi alpha with only 5 nonconservative amino acid differences. G proteins previously termed Gi alpha may fall into at least two distinct groups, with one including human myeloid Gi alpha, rat brain Gi alpha and mouse macrophage Gi alpha; and other Gx alpha and bovine brain Gi alpha. One group probably contains true Gi and the other a new class of G protein whose function remains to be determined.     

Gamma-subunits of G proteins, but not their alpha- or beta-subunits, are polyisoprenylated. Studies on post-translational modifications using in vitro translation with rabbit reticulocyte lysates

Lipid modifications that may be introduced into several subunits of G proteins were explored by in vitro translation of recombinant mRNAs in reticulocyte lysates. In agreement with studies by others, myristic acid was incorporated into alpha i's and alpha o, but not alpha s, beta, or gamma's. In contrast, mevalonate (Mev) was incorporated only into gamma-subunits. Both, the gamma-subunit of transducin (gamma T) and that of other G proteins (gamma G) were modified by the lysates but with different characteristics. Labeled gamma T was unstable and was rapidly proteolyzed. Labeled gamma G was stable. The Mev-derivative in gamma G was sensitive to methyliodide and, after cleavage and chromatographic analysis, comigrated with the C20 polyisoprenol geranylgeraniol. This indicated that gamma G had been geranylgeranylated and that this polyisoprenoid was attached to the protein through a thioether linkage. It is thought that polyisoprenylation is defined by the COOH-terminal sequence Cys-A-A-X, where A is an aliphatic acid and X is any amino acid. Replacement by mutation of the Cys of the COOH-terminal -Cys-Ala-Ile-Leu sequence of gamma G with Ser abolished Mev incorporation, suggesting this Cys as the site of attachment of the geranylgeranyl moiety. Yet, Mev incorporation was less than 10% as much into gamma G with the Cys-A-A-X sequence -Cys-Ala-Ile-Trp. Consistent with geranylgeranylation, the C15 farnesyl moiety of farnesyl pyrophosphate was not incorporated into gamma G unless the incubations were fortified with Mev. In contrast, the farnesyl moiety was incorporated in an Mev-independent manner into gamma T (COOH terminus: -Cys-Val-Ile-Ser) and c-Ha-ras (COOH terminus: -Cys-Val-Leu-Ser) which are both farnesylated rather than geranylgeranylated. Thus, 1) separate enzymes appear to be involved in transferring farnesyl and geranylgeranyl groups to proteins, 2) structural factors other than the CAAX box contribute to the activity of the polyisoprenylating enzymes, and 3) this type of lipidation may be part of a proteolytic signaling system. Polyisoprenylation, which increases hydrophobicity of the derivatized protein, may play a role in anchoring not only ras but also G proteins to membranes.     

Posttranslational modification of proteins by isoprenoids in mammalian cells

Isoprenylation is a posttranslational modification that involves the formation of thioether bonds between cysteine and isoprenyl groups derived from pyrophosphate intermediates of the cholesterol biosynthetic pathway. Numerous isoprenylated proteins have been detected in mammalian cells. Those identified include K-, N-, and H-p21ras, ras-related GTP-binding proteins such as G25K (Gp), nuclear lamin B and prelamin A, and the gamma subunits of heterotrimeric G proteins. The modified cysteine is located in the fourth position from the carboxyl terminus in every protein where this has been studied. For p21ras, the last three amino acids are subsequently removed and the exposed cysteine is carboxylmethylated. Similar processing events may occur in lamin B and G protein gamma subunits, but the proteolytic cleavage in prelamin A occurs upstream from the modified cysteine. Lamin B and p21ras are modified by C15 farnesyl groups, whereas other proteins such as the G protein gamma subunits are modified by C20 geranylgeranyl chains. Separate enzymes may catalyze these modifications. The structural features that govern the ability of particular proteins to serve as substrates for isoprenylation by C15 or C20 groups are not completely defined, but studies of the p21ras modification using purified farnesyl:protein transferase suggest that the sequence of the carboxyl-terminal tetrapeptide is important. Isoprenylation plays a critical role in promoting the association of p21ras and the lamins with the cell membrane and nuclear envelope, respectively. Future studies of the role of isoprenylation in the localization and function of ras-related GTP-binding proteins and signal-transducing G proteins should provide valuable new insight into the link between isoprenoid biosynthesis and cell growth.     

Purification of heterotrimeric GTP-binding proteins from brain: identification of a novel form of Go

Using high-resolution Mono-Q anion-exchange chromatography, we purified four distinct GTP-binding proteins from bovine brain. Each consists of alpha and associated beta/gamma subunits, and each is a substrate for pertussis toxin catalyzed ADP-ribosylation. We defined the relationship between the alpha subunits of the purified proteins and cloned cDNAs encoding putative alpha subunits (1) by performing immunoblots with peptide antisera with defined specificity and (2) by comparing the migration on two-dimensional gel electrophoresis of the purified proteins, and of the in vitro translated products of cDNAs encoding alpha subunits. Purified G proteins with alpha subunits of 39, 41, and 40 kDa (G39, G41, and G40 in order of abundance) correspond to the products of Go, Gi1, and Gi2 cDNAs. We purified a novel G protein with an alpha subunit slightly above 39 kDa (G39*). G39* is less abundant than G39, elutes earlier than G39 on Mono-Q chromatography, and has a more basic pI (6.0 vs 5.6) than G39. G39 and G39*, however, are indistinguishable on immunoblots with a large number of specific antisera. The data suggest that G39* may represent a novel form of Go, differing in posttranslational modification rather than primary sequence.     

Polyisoprenylation of the CAAX motif--an in vitro protein synthesis study

A number of proteins, including most nuclear lamins, certain fungal mating pheromones, G-protein gamma-subunits and ras proteins, contain a C-terminal cysteine-aliphatic-aliphatic-undefined amino acid (CAAX) motif which is thought to be a roughly defined consensus sequence capable of directing a series of posttranslational events, beginning with the addition of a polyisoprene moiety to the cysteine. So far such a motif has been found in every protein known to have this type of modification. We have utilized the rabbit reticulocyte lysate translation system, which is capable of carrying out the polyisoprene modification in vitro, to investigate features of the C-terminal motif which affect its suitability as a substrate. We demonstrate that a cysteine is only isoprenylated when situated at position -4 from the C-terminus. We further show that the presence of a glycine at position -3 or a terminal aromatic residue, features typical of some G-protein alpha subunits, cause a reduction and abolition respectively of isoprenylation.     

Identification of the subunits of GTP-binding proteins coupled to somatostatin receptors.

Somatostatin (SRIF) induces its biological effects by interacting with membrane-bound receptors that are linked to cellular effector systems via G proteins. We have studied SRIF receptor-G protein associations by solubilizing the SRIF receptor from rat brain and AtT-20 cells and immunoprecipitating the receptor-G protein complex with peptide-directed antisera against the different subunits of the G protein heterotrimer. Antiserum 8730, which selectively interacts with all Gi alpha subtypes, maximally and specifically immunoprecipitated SRIF receptor-Gi alpha complexes. To identify the subtypes of Gi alpha that are coupled to SRIF receptors, the subtype-selective antisera 3646, 1521, and 1518, which specifically interact with Gi alpha 1, Gi alpha 2, and Gi alpha 3, respectively, were used to immunoprecipitate SRIF receptor-Gi alpha complexes. Antiserum 3646 immunoprecipitated SRIF receptor-Gi alpha 1 complexes from both brain and AtT-20 cells. Antiserum 1521 immunoprecipitated Gi alpha 2 from both brain and AtT-20 cells but did not immunoprecipitate SRIF receptors from these tissues. Antiserum 1518 immunoprecipitated AtT-20 cell SRIF receptors but uncoupled brain SRIF receptor-G protein complexes. This result was confirmed with another peptide-selective antiserum, SQ, directed against Gi alpha 3. The findings from these studies indicate that Gi alpha 1 and Gi alpha 3 are coupled to SRIF receptors, whereas Gi alpha 2 is not. Even though brain and AtT-20 cell SRIF receptors were both coupled to Gi alpha, the receptors from these tissues differed in their coupling to Go alpha. Antiserum 2353, which is directed against Go alpha, immunoprecipitated SRIF receptors from AtT-20 cells, but did not immunoprecipitate or uncouple SRIF receptor-G protein complexes from rat brain. To determine the beta subunits associated with the SRIF receptor, antisera directed against G beta 36 and G beta 35 were used to immunoprecipitate SRIF receptor-G protein complexes from brain. Peptide-directed antiserum against G beta 36 selectively immunoprecipitated solubilized brain SRIF receptors. However, antiserum directed against the G beta 35 subunit did not immunoprecipitate brain SRIF receptors, suggesting that brain SRIF receptors may preferentially associate with G beta 36. In addition to coimmunoprecipitating with Gi alpha and G beta, brain SRIF receptors coimmunoprecipitated the G protein gamma subunits, G gamma 2 and G gamma 3. These results provide the first evidence that SRIF receptors are coupled to different subunits of G proteins and suggest that selectivity exists in the association of different G protein subunits with the SRIF receptor.     

The NH2-terminal alpha subunit attenuator domain confers regulation of G protein activation by beta gamma complexes.

Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the alpha s or alpha i subunit. The beta gamma subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and alpha subunit activation by GTP. Multiple beta and gamma polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The beta gamma complex from retinal rod outer segments (beta gamma t) has been shown to discriminate between alpha i and alpha s subunits (Helman et al: Eur J Biochem 169:431-439, 1987). beta gamma t efficiently interacts with alpha i-like G protein subunits, but poorly recognizes the alpha s subunit. beta gamma t was, therefore, used to define regions of the alpha i subunit polypeptide that conferred selective regulation compared to the alpha s polypeptide. A series of alpha subunit chimeras having NH2-terminal alpha i and COOH-terminal alpha s sequences were characterized for their regulation by beta gamma t, measured by the kinetics of GTP gamma S activation of adenylyl cyclase. A 122 amino acid NH2-terminal region of the alpha i polypeptide encoded within an alpha i/alpha s chimera was sufficient for beta gamma t to discriminate the chimera from alpha s. A shorter 54 amino acid alpha i sequence substituted for the corresponding NH2-terminal region of alpha s was insufficient to support the alpha i-like interaction with beta gamma t. The findings are consistent with our previous observation (Osawa et al: Cell 63:697-706, 1990) that a region in the NH2-terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the alpha subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by beta gamma complexes.     

Heterotrimer formation,together with isoprenylation,is required for plasma membrane targeting of Gbetagamma

Nascent beta and gamma subunits of heterotrimeric G proteins need to be targeted to the cytoplasmic face of the plasma membrane (PM) in order to transmit signals. We show that beta(1)gamma(2) is poorly targeted to the PM and predominantly localized to endoplasmic reticulum (ER) membranes when expressed in HEK293 cells, but co-expression of a G protein alpha subunit allows strong PM localization of the beta(1)gamma(2). Furthermore, C-terminal isoprenylation of the gamma subunit is necessary but not sufficient for PM localization of beta(1)gamma(2). Isoprenylation of gamma(2) and localization of beta(1)gamma(2) to the ER occurs independently of alpha expression. Efficient PM localization of beta(1)gamma(2) in the absence of co-expressed alpha is observed when a site for palmitoylation, a putative second membrane targeting signal, is introduced into gamma(2). When a mutant of alpha(s) is targeted to mitochondria, beta(1)gamma(2) follows, consistent with an important role for alpha in promoting subcellular localization of betagamma. Furthermore, we directly demonstrate the requirement for alpha by showing that disruption of heterotrimer formation by the introduction of alpha binding mutations into beta(1) impedes PM targeting of beta(1)gamma(2). The results indicate that two membrane targeting signals, lipid modification and alpha binding, make concerted contributions to PM localization of betagamma.     

Gbetagamma affinity for bovine rhodopsin is determined by the carboxyl-terminal sequences of the gamma subunit

Two native betagamma dimers, beta(1)gamma(1) and beta(1)gamma(2), display very different affinities for receptors. Since these gamma subunits differ in both primary structure and isoprenoid modification, we examined the relative contributions of each to Gbetagamma interaction with receptors. We constructed baculoviruses encoding gamma(1) and gamma(2) subunits with altered CAAX (where A is an aliphatic amino acid) motifs to direct alternate or no prenylation of the gamma chains and a set of gamma(1) and gamma(2) chimeras with the gamma(2) CAAX motif at the carboxyl terminus. All the gamma constructs coexpressed with beta(1) in Sf9 cells yielded beta(1)gamma dimers, which were purified to near homogeneity, and their affinities for receptors and Galpha were quantitatively determined. Whereas alteration of the isoprenoid of gamma(1) from farnesyl to geranylgeranyl and of gamma(2) from geranylgeranyl to farnesyl had no impact on the affinities of beta(1)gamma dimers for Galpha(t), the non-prenylated beta(1)gamma(2) dimer had significantly diminished affinity. Altered prenylation resulted in a <2-fold decrease in affinity of the beta(1)gamma(2) dimer for rhodopsin and a <3-fold change for the beta(1)gamma(1) dimer. In each case with identical isoprenylation, the beta(1)gamma(2) dimer displayed significantly greater affinity for rhodopsin compared with the beta(1)gamma(1) dimer. Furthermore, dimers containing chimeric Ggamma chains with identical geranylgeranyl modification displayed rhodopsin affinities largely determined by the carboxyl-terminal one-third of the protein. These results indicate that isoprenoid modification of the Ggamma subunit is essential for binding to both Galpha and receptors. The isoprenoid type influences the binding affinity for receptors, but not for Galpha. Finally, the primary structure of the Ggamma subunit provides a major contribution to receptor binding of Gbetagamma, with the carboxyl-terminal sequence conferring receptor selectivity.     

Location of signal sequences for membrane insertion of the Na+,K+-ATPase alpha subunit.

To study the membrane insertion of the Na+,K+-ATPase (EC 3.6.1.37) alpha subunit with six to eight transmembrane segments, mRNAs encoding the entire alpha subunit and its four different domains were prepared and translated in rabbit reticulocyte lysate with rough microsomal membranes. On the basis of the resistance of the membrane-inserted products to alkali extraction and the failure to insert the translation products into N-ethylmaleimide-treated membranes, it is suggested that at least two signal sequences are contained within the four transmembrane segments from the amino terminus of the alpha subunit.     

Differential sensitivity of phosphatidylinositol 3-kinase p110gamma to isoforms of G protein betagamma dimers

The ability of G protein alpha and betagamma subunits to activate the p110gamma isoform of phosphatidylinositol 3-kinase (PtdIns 3-kinase) was examined using pure, recombinant G proteins and the p101/p110gamma form of PtdIns 3-kinase reconstituted into synthetic lipid vesicles. GTP-activated Gs, Gi, Gq, or Go alpha subunits were unable to activate PtdIns 3-kinase. Dimers containing Gbeta(1-4) complexed with gamma2-stimulated PtdIns 3-kinase activity about 26-fold with EC50 values ranging from 4 to 7 nm. Gbeta5gamma2 was not able to stimulate PtdIns 3-kinase despite producing a 10-fold activation of avian phospholipase Cbeta. A series of dimers with beta subunits containing point mutations in the amino acids that undergo a conformational change upon interaction of betagamma with phosducin (beta1H311Agamma2, beta1R314Agamma2, and beta1W332Agamma2) was tested, and only beta1W332Agamma2 inhibited the ability of the dimer to stimulate PtdIns 3-kinase. Dimers containing the beta1 subunit complexed with a panel of different Ggamma subunits displayed variation in their ability to stimulate PtdIns 3-kinase. The beta1gamma2, beta1gamma10, beta1gamma12, and beta1gamma13 dimers all activated PtdIns 3-kinase about 26-fold with 4-25 nm EC50 values. The beta1gamma11 dimer, which contains the farnesyl isoprenoid group and is highly expressed in tissues containing the p101/p110gamma form of PtdIns 3-kinase, was ineffective. The role of the prenyl group on the gamma subunit in determining the activation of PtdIns 3-kinase was examined using gamma subunits with altered CAAX boxes directing the addition of farnesyl to the gamma2 subunit and geranylgeranyl to the gamma1 and gamma11 subunits. Replacement of the geranylgeranyl group of the gamma2 subunit with farnesyl inhibited the activity of beta1gamma2 on PtdIns 3-kinase. Conversely, replacement of the farnesyl group on the gamma1 and gamma11 subunit with geranylgeranyl restored almost full activity. These findings suggest that all beta subunits, with the exception of beta5, interact equally well with PtdIns 3-kinase. In contrast, the composition of the gamma subunit and its prenyl group markedly affects the ability of the betagamma dimer to stimulate PtdIns 3-kinase.     

The posttranslationally modified C-terminal structure of bovine aortic smooth muscle rhoA p21

rhoA p21, a ras p21-like small GTP-binding protein, has the same C-terminal consensus motif of Cys-A-A-X (A is an aliphatic amino acid and X is any amino acid) as ras p21s, which is posttranslationally processed. We here determine the posttranslationally processed C-terminal structure of the rhoA p21 purified from bovine aortic smooth muscle. Incubation of rhoA p21-expressing insect cells with exogenous [3H]mevalonolactone caused the labeling of rhoA p21, suggesting that rhoA p21 is prenylated. Consistently, Raney nickel treatment of rhoA p21 released a geranylgeranyl moiety as estimated by gas chromatography/mass spectrometry. No lipid moiety was released by KOH or NH2OH treatment. Extensive digestion of rhoA p21 with Achromobacter protease I yielded a C-terminal peptide, Ser-Gly-Cys190, that lacked the three C-terminal amino acids predicted from the cDNA but was geranylgeranylated and carboxyl methylated at the cysteine residue. Bovine brain cytosol geranylgeranylated the bacterial rhoA p21 having the three C-terminal amino acids predicted from the cDNA but not the protein lacking the three C-terminal amino acids. Bovine brain membranes methylated the synthetic C-terminal peptide with 10 amino acids of rhoA p21 which was geranylgeranylated at its C-terminal cysteine residue but not the peptide which was not geranylgeranylated. These results suggest that rhoA p21 is first geranylgeranylated followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the exposed cysteine residue.     

Reconstitution reveals additional roles for N- and C-terminal domains of g(alpha) in muscarinic receptor coupling

The molecular basis for selectivity of M1 and M2 muscarinic receptor coupling to heterotrimeric G proteins has been studied using receptors expressed in Sf9 cell membranes and reconstituted with purified chimeric G(alpha) subunits containing different regions of Gi1alpha and Gq(alpha). The abilities of G protein heterotrimers containing chimeric alpha subunits to stabilize the high-affinity state of the receptors for agonist and to undergo receptor stimulated guanine nucleotide exchange was compared with G protein heterotrimers containing either native Gi1alpha or Gq(alpha). The data confirm the importance of the proper context of the C-terminus of Galpha by demonstrating that the C-terminus of Gi1alpha, when placed in the context of Gq(alpha), prevents coupling to muscarinic M1 receptors, while the C-terminus of Gq(alpha), when placed in the context of Gi1alpha, prevents coupling to muscarinic M2 receptors. However, C-terminal amino acids of Gq(alpha) placed in the context of Gi1alpha were not sufficient to allow M1 receptor coupling, nor were C-terminal amino acids of Gi1alpha placed in the context of Gq(alpha) sufficient for M2 receptor coupling. The unique six amino acid N-terminal extension of Gq(alpha) when added to the N-terminus of Gi1alpha neither prevented M2 receptor coupling nor permitted M1 receptor coupling. A Gi1alpha-based chimera containing both N- and C-terminal regions of Gq(alpha) gained the ability to productively couple M1 receptors suggesting that the proper context of both N- and C-termini is required for muscarinic receptor coupling.