Selective reconstitution of gastrin-releasing peptide receptor with G alpha q

Identification of the molecular mechanisms that determine specificity of coupling interactions between gastrin-releasing peptide receptors (GRPrs) and their cognate heterotrimeric GTP-binding proteins is a fundamental step in understanding the signal transduction cascade initiated by receptor-ligand interaction. To explore these mechanisms in greater detail, we have developed an in situ reconstitution assay in chaotrope-extracted membranes from mouse fibroblasts expressing the GRPr, and we have used it to measure GRPr-catalyzed binding of GTP gamma S to purified G protein alpha subunits. Binding studies with 125I-labeled [D-Tyr6]bombesin(6-13) methyl ester (125I-Tyr-ME), a GRPr specific antagonist, show a single binding site with a Kd = 1.4 nM +/- 0.4 (mean +/- SD, n = 3) and capacity of 15-22 pmol of receptor per mg of protein in the extracted membrane preparations, representing a 2- to 3-fold enrichment of binding sites compared with the membranes before extraction. Quantitative ligand displacement analysis using various unlabeled GRPr agonists shows a rank order of potency characteristic of the GRPr: bombesin > or = GRP > > neuromedin B. Reconstitution of urea extracted membranes with a purified G alpha q showed that receptor-catalyzed binding of GTP gamma S was dependent on agonist (GRP) and G beta gamma subunits. The EC50 for GRP was 3.5 nM, which correlates well with the reported Kd of 3.1 nM for GRP binding to GRPr expressed in mouse fibroblasts [Benya, R. V., et al. (1994) Mol. Pharmacol. 46, 235-245]. The apparent Kd for bovine brain G beta gamma in this assay was 60 nM, and the Km for squid retinal G alpha q was 90 nM. The GRPr-catalyzed binding of GTP gamma S is selective for G alpha q, since we did not detect receptor-catalyzed exchange using either G alpha i/o or G alpha t. These data demonstrate that GRPr can functionally couple to G alpha q but not to the pertussis toxin-sensitive G alpha i/o or retinal specific G alpha t. This in situ receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.     

Expression and characterization of the rat D3 dopamine receptor: pharmacologic properties and development of antibodies

Serotonin (5-HT) potently contracts the fundus of the rat stomach; however, the associated transduction pathway has not been described fully. Experiments were performed in an attempt to gain insight into the coupling mechanism associated with this fundal 5-HT receptor. 5-HT-stimulated [35S]GTP gamma S binding to a protein which was recognized by anti-G alpha Z antiserum in a Mg(++)-dependent fashion. 5-HT increased [35S]GTP gamma S binding in the fundus, but not in the corpus of the rat stomach. 5-HT also enhanced the binding of [alpha-32P]GTP to the fundal protein and increased the hydrolysis of GTP to GDP in fundal membranes. The fundal protein which binds GTP is 25 to 29 kDa in size whereas the brain G alpha Z protein which is recognized by the anti-G alpha Z antibody is a 41 kDa protein. Mixing experiments revealed that the fundal guanine nucleotide binding protein does not appear to be a proteolytic product of the 41 kDa G alpha Z protein. Activating protein kinase C with phorbol-12-myristate, 13-acetate induced a concentration-dependent, noncompetitive inhibition of [35S]GTP gamma S binding to the fundal protein, and of 5-HT-induced contraction of fundal strips. Phorbol-12-myristate, 13-acetate did not alter carbachol- or KCl-mediated fundus contraction. Furthermore, the activation of [35S]GTP gamma S binding by serotonergic agonists and its inhibition by pharmacological antagonists corresponded to the known actions of these agents on contraction of fundal muscle. The results provide evidence that the 5-HT receptor in the rat stomach fundus is coupled directly or indirectly to a G alpha z-like protein which may mediate 5-HT-induced contraction in this tissue.     

Identification of a distinct molecular mass G alpha(h) (transglutaminase II) coupled to alpha1-adrenoceptor in mouse heart

Our previous studies on alpha1-adrenoceptor signaling suggested that G alpha(h) family is a signal mediator in different species. To elucidate the species-specificity of G alpha(h) family in molecular mass, we used the solubilized membranes from mouse heart and the ternary complex preparations containing alpha1-agonist/receptor/G-protein. Binding of [35S]GTPgammaS and the intensity of the [alpha-32P]GTP photoaffinity labeled protein resulting from activation of the alpha1-adrenoceptor were significantly attenuated by the antagonist, phentolamine. The molecular mass of the specific GTP-binding protein was approximately 72-kDa; homologous with G alpha(h) (transglutaminase II) family. Furthermore, immunological cross-reactivity of ternary complex from mouse heart and purified G alpha(h) from rat, guinea pig, and bovine using anti-G alpha(h7) antibody showed that their molecular masses were distinctly different and approximately 72-kDa G alpha(h) from mouse heart was the lowest molecular mass. Consistent with these observations, in co-immunoprecipitation and co-immunoadsorption of the alpha1-adrenoceptor in the ternary complex preparation by anti-G alpha(h7) antibody, the G alpha(h) family protein tightly coupled to alpha1-adrenoceptor. These results demonstrate the species-specificity of G alpha(h) family in molecular mass, especially the lowest molecular mass in mouse.     

Identification of a structural element in phospholipase C beta2 that interacts with G protein betagamma subunits

To delineate the specific regions of phospholipase C beta2 (PLC beta2) involved in binding and activation by G protein betagamma subunits, we synthesized peptides corresponding to segments of PLC beta2. Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC beta2 by betagamma subunits (IC50 50 and 150 microM, respectively), whereas two control peptides did not. N20K and E20K, but not the control peptides, inhibited betagamma-dependent ADP-ribosylation of Galphai1 by pertussis toxin and betagamma-dependent activation of phosphoinositide 3-kinase. To demonstrate direct binding of the peptides to betagamma subunits, the peptides were chemically cross-linked to purified beta1gamma2. N20K and E20K cross-linked to both beta1 and gamma2 subunits, whereas the control peptides did not. Cross-linking to beta and gamma was inhibited by incubation with excess PLC beta2 or PLC beta3, whereas cross-linking to gamma but not beta was inhibited by r-myr-alphai1. These data together demonstrate specificity of N20K and E20K for G betagamma binding and inhibition of effector activation by betagamma subunits. The results suggest that an overlapping region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC beta2 that is involved in binding to betagamma subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to betagamma subunits. Alignment of these peptides with the three-dimensional structure from PLC delta1 identifies a putative alpha helical region on the surface of the catalytic domain of PLC beta2 that could interact with betagamma subunits.     

Phosphatidyl inositol-phospholipase C in squid photoreceptor membrane is activated by stable metarhodopsin via GTP-binding protein, Gq

Phosphatidyl inositol-phospholipase C (PI-PLC) in squid retina was studied by immunoblotting and its activities were determined using [3H]phosphatidyl inositol bisphosphate ([3H]PIP2) as substrate. PI-PLC activity was found mostly in soluble fraction when the retina homogenate was treated with 400 mM KCl, but was associated with rhabdomal membranes under low salt conditions (20 mM Hepes). A protein with apparent molecular mass of 130kD was recognized by an antibody against PLC beta 4/norp A in both 400 mM KCl soluble and rhabdomal membrane fractions. A 42 kD protein recognized by antibody against the C-terminus of Gq alpha was also present in these two fractions. GTP gamma S stimulated only the PI-PLC activity associated with membrane and was magnesium dependent. PI-PLC activity was found to be (i) highly dependent upon calcium concentrations, (ii) enhanced by GTP but not by other nucleotides, and (iii) significantly stimulated by light at lower concentrations of GTP gamma S. The stimulation by light was still observed when irradiated membrane was incubated at 10 degrees C for 10 min and then mixed with GTP gamma S. These results suggest that stable metarhodopsin stimulates a PLC beta 4/norp A-like enzyme via a G-protein, Gq.     

Antioxidants and inflammatory cell response in preeclampsia

We have previously reported that gastrin induces a rapid and transient tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1) in association with inositol 1,4,5-trisphosphate (IP3) formation in rat colonic epithelial cells (34). In this study, we demonstrate that gastrin regulates IP3 formation mainly through PLC gamma 1 isozyme. Immunoblotting analysis revealed the expression of PLC beta 3 and -gamma 1, but not PLC beta 1, -beta 2, or -beta 4 in the rat colonic epitheliums. To explore what PLC isozyme(s) modulates gastrin effect on IP3, immunoneutralizing antibody to PLC beta 1, -beta 3, or -gamma 1 was introduced into the colonic cells using a lipid carrier. The gastrin-stimulated increase in IP3 concentration was specifically prevented by anti-PLC gamma 1 but not by anti-PLC beta 1 or -beta 3 antibody. Immunoprecipitation assays have also revealed that gastrin promoted an increase in tyrosine phosphorylation and co-precipitation of a 60 kDa src kinase with PLC gamma 1. Administration of antibody specific to pp60c-src into the colonic cells prevented the gastrin-stimulated increases in IP3. Tyrosine phosphorylation of PLC gamma 1 may be a major mechanism through which gastrin regulates IP3 level in the colonic cells. Pretreatment of cells with the tyrosine kinase inhibitor genistein abrogated gastrin's effect on IP3, while extended pretreatment with pertussis toxin, a G-protein inhibitor, did not affect the ability of gastrin to stimulate IP3 formation. Colonic cells expressed the G alpha i subunits1-3; however, immunoblotting analysis did not reveal any difference in G alpha i proteins' expression between control and gastrin treated cells. The results provide direct evidence that gastrin regulates IP3 level by a signaling mechanism that involves PLC gamma 1 and pp60c-src kinase.     

Functional coupling of NKR-P1 receptors to various heterotrimeric G proteins in rat interleukin-2-activated natural killer cells

NKR-P1 molecules constitute a family of type II membrane receptors in natural killer (NK) cells that preferentially activate NK cell killing and release of interferon-gamma from these cells. Here, we demonstrate that anti-NKR-P1 enhances GTP binding in rat interleukin-2-activated NK cell membranes; GTP binding to Gi3alpha, Gsalpha, Gq,11alpha, and Gzalpha increased noticeably in these cell membranes after treatment with anti-NKR-P1. Western blot analysis of membrane proteins prepared from interleukin-2-activated NK cells reveals the presence of Gi1,2alpha, Gi3alpha, Goalpha, Gsalpha, Gq, 11alpha, Gzalpha, and G12alpha, but not G13alpha. However, only alphai3, alphas, alphaq,11, and alphaz, but not alphai1,2, alphao, alpha12, or alpha13 subunits when immunoprecipitated with the appropriate anti-G protein antibodies, are associated with NKR-P1 when immunoblotted with anti-NKR-P1. Reciprocally, NKR-P1 immunoprecipitated with anti-NKR-P1 is associated with alphai3, alphas, alphaq,11, and alphaz immunoblotted with anti-G proteins. These results are the first to demonstrate the physical and functional coupling of NKR-P1 to the heterotrimeric G proteins in NK cells.     

Purification and characterization of a guanine nucleotide-exchange protein for ADP-ribosylation factor from spleen cytosol

ADP-ribosylation factors (ARFs) are 20-kDa guanine nucleotide-binding proteins and are active in the GTP-bound state and inactive with GDP bound. ARF-GTP has a critical role in vesicular transport in several cellular compartments. Conversion of ARF-GDP to ARF-GTP is promoted by a guanine nucleotide-exchange protein (GEP). We earlier reported the isolation from bovine brain cytosol of a 700-kDa protein complex containing GEP activity that was inhibited by brefeldin A (BFA). Partial purification yielded an approximately 60-kDa BFA-insensitive GEP that enhanced binding of ARF1 and ARF3 to Golgi membranes. GEP has now been purified extensively from rat spleen cytosol in a BFA-insensitive, approximately 55-kDa form. It activated class I ARFs (ARFs 1 and 3) that were N-terminally myristoylated, but not nonmyristoylated ARFs from class-I, II, or III. GEP activity required MgCl2. In the presence of 0.6-0.8 mM MgCl2 and 1 mM EDTA, binding of guanosine 5'-[gamma[35S]thio]triphosphate ([35S]GTP gamma S) by ARF1 and ARF3 was equally high without and with GEP. At higher Mg2+ concentrations, binding without GEP was much lower; with 2-5 mM MgCl2, GEP-stimulated binding was maximal. The rate of GDP binding was much less than that of GTP gamma S with and without GEP. Phospholipids were necessary for GEP activity; phosphatidylinositol was more effective than phosphatidylserine, and phosphatidic acid was less so. Other phospholipids tested were ineffective. Maximal effects required approximately 200 microM phospholipid, with half-maximal activation at 15-20 microM. Release of bound [35S]GTP gamma S from ARF3 required the presence of both GEP and unlabeled GTP or GTP gamma S; GDP was much less effective. This characterization of the striking effects of Mg2+ concentration and specific phospholipids on the purified BFA-insensitive ARF GEP should facilitate experiments to define its function in vesicular transport.     

Purification of four forms of the beta gamma subunit complex of G proteins containing different gamma subunits

To investigate the physiological significance of the diversity of gamma subunits of G proteins, we purified four forms of beta gamma of G proteins from bovine brain (beta gamma-B1, beta gamma-B2, beta gamma-B3), and spleen (beta gamma-S1) by the sequential chromatography on columns of DEAE-Sephacel, Ultrogel AcA 34, heptylamine-Sepharose, phenyl-5PW, and DEAE-5PW. Electrophoretic analyses showed that each beta gamma mainly contained the 36-kDa beta and a distinct but homogeneous gamma. These beta gamma complexes were subjected directly to proteolytic digestion and subsequent amino acid sequence analyses of their fragments. It was revealed that beta gamma-B1, -B2, and -B3 were identical to beta 1 gamma 7 (with a low level of beta 2 gamma 7), beta 1 gamma 2 and beta 1 gamma 3, respectively, while beta gamma-S1 was composed of beta 1 and an unidentified form of gamma. Then we examined the functional differences among these beta gamma complexes and the beta gamma of transducin (beta gamma-T, beta 1 gamma 1). Few differences were observed among all beta gamma complexes to enhance pertussis toxin-catalyzed ADP-ribosylation of the alpha subunits of G(o) and Gt. The four forms of beta gamma complexes purified from brain and spleen showed indistinguishable inhibitory effects on the release of GDP from G(o) alpha, but beta gamma-T was much less effective. Brain and spleen beta gamma complexes were equally effective in inhibiting calmodulin-stimulated adenylyl-cyclase activity, but beta gamma-T had a very weak inhibitory effect. Five forms of beta gamma facilitated metarhodopsin II-catalyzed binding of GTP gamma S to Gt alpha in a concentration-dependent manner with the following rank order of effectiveness: beta gamma-S1 > beta gamma-T > beta gamma-B1 > beta gamma-B2 > beta gamma-B3. Because the beta gamma complexes used in this study mostly contained the same beta subunit, the functional differences must be dependent on the gamma subunits. Thus, it seems likely that the receptor, the alpha subunits, and the effector are able to distinguish between the various gamma subunits.     

Reconstitution of beta2-adrenoceptor-GTP-binding-protein interaction in Sf9 cells--high coupling efficiency in a beta2-adrenoceptor-G(s alpha) fusion protein

In most studies, coupling of the beta2-adrenoceptor (beta2AR) to the stimulatory, heterotrimeric GTP-binding protein of adenylyl cyclase the (Gs) is studied indirectly by measuring adenylyl cyclase activation. The aim of this study was to establish a model system in which beta2AR-Gs interactions can be studied directly at the level of the G-protein. We expressed the beta2AR alone, in combination with the alpha-subunit of Gs (G(s alpha)), and as fusion protein with G(s alpha) (beta2AR-G(s alpha)) in Sf9 insect cells. The beta2AR expressed alone couples poorly to the endogenous G(s alpha)-like G-protein of Sf9 cells since no high-affinity agonist binding could be detected, and the effects of agonist and inverse agonist on adenylyl cyclase, high-affinity GTPase and guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding were small. Beta2AR-G(s alpha) reconstituted high-affinity agonist binding and regulated adenylyl cyclase more effectively than the beta2AR co-expressed with a large excess of G(s alpha). In membranes expressing beta2AR-G(s alpha), highly effective agonist- and inverse agonist regulation of high-affinity GTP hydrolysis and GTP[S] binding was observed. In contrast, agonist and inverse agonist regulation of GTP hydrolysis and GTP[S] binding in membranes expressing beta2AR and G(s alpha) as separate proteins was difficult to detect. Our data show that the beta2AR interacts with G(s alpha) more efficiently when expressed as a fusion protein than when expressed with an excess of non-fused G(s alpha). The beta2AR-G(s alpha) fusion protein provides a very sensitive model system to study the regulation of Gs function by beta2AR agonists and inverse agonists directly at the level of the G-protein.     

Binding of G protein beta gamma-subunits to pleckstrin homology domains

Ligand-induced activation of many receptors leads to dissociation of the alpha- and beta gamma-subunit complexes of heterotrimeric G proteins, both of which regulate a variety of effector molecules involved in cellular signaling processes. In one case, a cytosolic enzyme, the beta-adrenergic receptor kinase (beta ARK) binds to the dissociated, prenylated, membrane-anchored beta gamma-subunits of heterotrimeric G proteins (G beta gamma) and is thereby targeted to its membrane-bound receptor substrate. Quite recently, numerous proteins involved in cellular signal transduction have been shown to contain sequences homologous with a "domain" originally identified in the protein "pleckstrin" (pleckstrin homology domain; PH domain) and subsequently found in the G beta gamma interaction region of the beta ARK sequence. Here we demonstrate that glutathione S-transferase-fusion proteins, containing sequences encompassing the PH domain of nine proteins from this group, bind G beta gamma to varying extents. Binding of G beta gamma to these fusion proteins was documented either by a direct binding assay or by ability to block G beta gamma-mediated membrane translocation of beta ARK1. G beta gamma binding to these fusion proteins was inhibited by the alpha subunit of Go (Go alpha), indicating that the binding of G beta gamma to G alpha and the PH domain-containing fusion proteins is mutually exclusive. Studies with a series of truncated PH domains derived from the Ras-guanine-nucleotide-releasing factor indicate that the G beta gamma binding domain includes only the C-terminal portion of the PH domain and sequences just distal to this. Protein-protein interactions between G beta gamma and PH domain-containing proteins may play a significant role in cellular signaling analogous to that previously demonstrated for Src homology 2 and 3 domains.     

RGS4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis

Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by alpha subunits of the Gi family but have not been reported to regulate other G protein alpha subunits. Expression of recombinant RGS proteins in cultured cells inhibits Gi-mediated hormonal signals probably by acting as GTPase-activating proteins for Galphai subunits. To ask whether an RGS protein can also regulate cellular responses mediated by G proteins in the Gq/11 family, we compared activation of mitogen-activated protein kinase (MAPK) by a Gq/11-coupled receptor, the bombesin receptor (BR), and a Gi-coupled receptor, the D2 dopamine receptor, transiently co-expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples Gi from receptors, blocked MAPK activation by the D2 dopamine receptor but not by the BR. Co-expression of RGS4, however, inhibited activation of MAPK by both receptors causing a rightward shift of the concentration-effect curve for both receptor agonists. RGS4 also inhibited BR-stimulated synthesis of inositol phosphates by an effector target of Gq/11, phospholipase C. Moreover, RGS4 inhibited inositol phosphate synthesis activated by addition of AlF4- to cells overexpressing recombinant alphaq, probably by binding to alphaq.GDP.AlF4-. These results demonstrate that RGS4 can regulate Gq/11-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein.     

Characterization of the histamine H2 receptor structural components involved in dual signaling

We previously demonstrated that the histamine H2 receptor can activate both adenylate cyclase (AC) and phospholipase C (PLC) signaling pathways via separate GTP-dependent mechanisms. We examined whether H2 receptor-specific peptides corresponding to the amino (N) or carboxyl terminus (C) of the second (2i) or third (3i) intracytoplasmic loops or the carboxyl terminal tail (P4iN) could effect histamine- stimulated AC and PLC activity in cell membranes prepared from HEPA cells stably transfected to express the canine H2 histamine receptor cDNA. Tiotidine binding and basal signaling were not altered by the synthetic peptides. H2P2iN, H2P2iC, H2P3iN and H2P4iN did not effect histamine stimulated AC activity although H2P3iC (10(-4) M) significantly inhibited this parameter (65.6 +/- 7.2% of maximal stimulation) (n = 6). Combination of the five peptides (H2P2iN, H2P2iC, H2P3iN, H2P3iC and H2P4iN) abolished histamine stimulated AC activity. Although all of the peptides inhibited histamine-stimulated PLC activity to a moderate degree individually, H2P3iC (10(-4) M) had the greatest effect, decreasing PLC activation to 20.8 +/- 6.3% of maximal stimulation (IC50 = 7.5 X 10(-7) M) (n = 6). H2P3iC and the peptide combination did not alter, forskolin, GTP gamma s or epinephrine-stimulated AC activity nor GTP gamma s and vasopressin-stimulated PLC. These studies demonstrate that both the second and third intracytoplasmic loops of the histamine H2 receptor are linked to separate signaling pathways in a differential manner.     

Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells

Members of the beta isozyme subfamily of phosphatidylinositol-specific phospholipase C (PLC) are stimulated by alpha subunits and betagamma dimers of heterotrimeric guanine-nucleotide-binding proteins (G proteins). Myeloid differentiated human HL-60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5'-->O)-3-thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCbeta2. The resolved factor, which behaved as 48-kDa protein upon gel filtration, stimulated PLCbeta2 but not PLCbeta1 or PLCdelta1. Activation of phosphatidylinositol 4-phosphate 5-kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCbeta2 that lacked a carboxyl-terminal region necessary for stimulation by members of the alpha(q) subfamily of the G-protein alpha subunits. The results of this study suggest that a GTP-binding protein distinct from alpha(q) subunits, probably a low-molecular-mass GTP-binding protein associated with a regulatory protein, is involved in isozyme-specific activation of PLCbeta2.     

Direct interaction of the alpha and gamma subunits of the G proteins. Purification and analysis by limited proteolysis

The heterotrimeric G proteins are often regarded functionally as a heterodimer, consisting of a guanine nucleotide-binding alpha subunit and a beta gamma subunit complex. Since the tightly associated beta gamma subunit complex can be separated only under denaturing conditions, studies aimed at determining the individual contributions of the beta and gamma subunits in terms of binding to the various alpha subunits, interacting with receptors, and regulating effectors, have not been possible. To circumvent this problem, we have used baculovirus-infected cells to direct the individual expression of the beta 1 and gamma 2 subunits. Application of extracts from baculovirus-infected cells to an alpha subunit of G protein (G(o) alpha)-affinity matrix resulted in the selective retention and AMF-specific elution of the expressed gamma 2 subunit, but not the expressed beta 1 subunit. Overall, these and other data provide the first evidence of a direct association between the gamma and alpha subunits, which is dependent on prenylation of gamma. The apparent direct association between the gamma and alpha subunits was further probed by limited trypsin proteolysis. Upon addition of trypsin, the G(o) alpha subunit was rapidly cleaved to a 24-kDa fragment. However, in the presence of the purified gamma 2 subunit, trypsin cleavage of the G(o) alpha subunit was completely prevented. This demonstration of a direct association between the gamma and alpha subunits is particularly intriguing in light of the increasingly large number of known alpha, beta, and gamma subunits, which raises important questions regarding the assembly of these subunits into functionally distinct G proteins. Thus, a direct association between the gamma and alpha subunits, which exhibit the greatest structural diversity, may provide the basis for the selective assembly of these subunits into G proteins with functional diversity.     

Identification of three isoforms for the Na+-dependent phosphate cotransporter (NaPi-2) in rat kidney

We have isolated three unique NaPi-2-related protein cDNAs (NaPi-2alpha, NaPi-2beta, and NaPi-2gamma) from a rat kidney library. NaPi-2alpha cDNA encodes 337 amino acids which have high homology to the N-terminal half of NaPi-2 containing 3 transmembrane domains. NaPi-2beta encodes 327 amino acids which are identical to the N-terminal region of NaPi-2 containing 4 transmembrane domains, whereas the 146 amino acids in the C-terminal region are completely different. In contrast, NaPi-2gamma encodes 268 amino acids which are identical to the C-terminal half of NaPi-2. An analysis of phage and cosmid clones indicated that the three related proteins were produced by alternative splicing in the NaPi-2 gene. In a rabbit reticulocyte lysate system, NaPi-2 alpha, beta, and gamma were found to be 36, 36, and 29 kDa amino acid polypeptides, respectively. NaPi-2alpha and NaPi-2gamma were glycosylated and revealed to be 45- and 35-kDa proteins, respectively. In isolated brush-border membrane vesicles, an N-terminal antibody was reacted with 45- and 40-kDa, and a C-terminal antibody was reacted with 37-kDa protein. The sizes of these proteins corresponded to those in glycosylated forms. A functional analysis demonstrated that NaPi-2gamma and -2alpha markedly inhibited NaPi-2 activity in Xenopus oocytes. The results suggest that these short isoforms may function as a dominant negative inhibitor of the full-length transporter.     

Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein alpha subunits and by the composition of the betagamma dimers. To determine the role of the gamma subunit prenyl modification in this interaction, the CaaX motifs in the gamma1 and gamma2 subunits were altered to direct modification with different prenyl groups, recombinant betagamma dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the betagamma dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the alpha subunit. The beta1gamma1 dimer (modified with farnesyl) was significantly less effective than beta1gamma2 (modified with geranylgeranyl) in either assay. The beta1gamma1-S74L dimer (modified with geranylgeranyl) was nearly as effective as beta1gamma2 in either assay. The beta1gamma2-L71S dimer (modified with farnesyl) was significantly less active than beta1gamma2. Using 125I-labeled betagamma subunits, it was determined that native and altered betagamma dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the gamma subunit is an important determinant of the interaction between receptors and G protein gamma subunits.     

Identification of integrin-dependent and -independent cell adhesion domains in COOH-terminal globular region of laminin-5 alpha 3 chain

Laminin-5 is an isoform of laminin that consists of alpha 3, beta 3, and gamma 2 chains and has potent cell adhesion- and cell migration-promoting activities. In this study, five subdomains in the COOH-terminal globular (G) domain of human laminin alpha 3 chain were individually expressed in Escherichia coli, and their biological activities were investigated. Recombinant G2, G4, and G5 domains promoted adhesion to plastic plates of HT1080 fibrosarcoma cells, A431 epidermoid carcinoma cells, and ECV304 vascular endothelial cells. For the cell adhesion activity, the G2 domain required a divalent cation and heat-sensitive conformation more strongly than G4 and G5. The cell adhesion to G2 but not G4 and G5 was effectively inhibited by an anti-integrin alpha 3 antibody. A cell adhesion sequence of 22 amino acids, alpha 3G2A, that was homologous to the integrin alpha 3 beta 1-binding sequence GD-6 of laminin alpha 1 chain was identified within the G2 structure. The cell adhesion to alpha 3G2A peptide was also inhibited by the anti-integrin alpha 3 antibody. The cell adhesion to G2, alpha 3G2A, G4, and G5 was strongly inhibited by heparin, but that to native laminin-5 was inhibited less effectively. Moreover, G5 potently stimulated chemotactic migration of rat liver epithelial cells in Boyden chambers, but G2 and G4 did not. These results indicate that the G domain of laminin alpha 3 contains multiple cell binding sites with different mechanisms and different functions. The G2 domain seems to recognize integrin alpha 3 beta 1, whereas G4 and G5 may interact with heparin-like molecules on cell surface.