Influence of differential stability of G protein βγ dimers containing the γ11 subunit on functional activity at the M1 muscarinic receptor, A1 adenosine receptor, and phospholipase C-β

Ggamma11 is an unusual guanine nucleotide-binding regulatory protein (G protein) subunit. To study the effect of different Gbeta-binding partners on gamma11 function, four recombinant betagamma dimers, beta1gamma2, beta4gamma2, beta1gamma11, and beta4gamma11, were characterized in a receptor reconstitution assay with the G(q)-linked M1 muscarinic and the G(i1)-linked A1 adenosine receptors. The beta4gamma11 dimer was up to 30-fold less efficient than beta4gamma2 at promoting agonist-dependent binding of [35S]GTPgammaS to either alpha(q) or alpha(i1). Using a competition assay to measure relative affinities of purified betagamma dimers for alpha, the beta4gamma11 dimer had a 15-fold lower affinity for G(i1) alpha than beta4gamma2. Chromatographic characterization of the beta4gamma11 dimer revealed that the betagamma is stable in a heterotrimeric complex with G(i1) alpha; however, upon activation of alpha with MgCl2 and GTPgammaS under nondenaturing conditions, the beta4 and gamma11 subunits dissociate. Activation of purified G(i1) alpha:beta4gamma11 with Mg+2/GTPgammaS following reconstitution into lipid vesicles and incubation with phospholipase C (PLC)-beta resulted in stimulation of PLC-beta activity; however, when this activation preceded reconstitution into vesicles, PLC-beta activity was markedly diminished. In a membrane coupling assay designed to measure the ability of G protein to promote a high-affinity agonist-binding conformation of the A1 adenosine receptor, beta4gamma11 was as effective as beta4gamma2 when coexpressed with G(i1) alpha and receptor. However, G(i1) alpha:beta4gamma11-induced high-affinity binding was up to 20-fold more sensitive to GTPgammaS than G(i1) alpha:beta4gamma2-induced high-affinity binding. These results suggest that the stability of the beta4gamma11 dimer can modulate G protein activity at the receptor and effector.     

Mutational effects at the subunit interfaces of human hemoglobin: evidence for a unique sensitivity of the T quaternary state to changes in the hinge region of the alpha 1 beta 2 interface

A set of variant human hemoglobins, each with an Ala or Gly substitution at a single residue, has been prepared, and the kinetics of their reactions with carbon monoxide have been measured. This reaction is rate-limited by the binding of the first CO to the deoxygenated T state of the protein. The magnitudes of the effects of the mutations on CO combination vary widely, and, with the exception of beta Y145, the residues with the most significant effects on these kinetics are found in the hinge region of the alpha 1 beta 2 interface. Mixed-metal hybrids, with zinc protoporphyrin IX in place of heme on both alpha or both beta subunits, were prepared for beta W37E, beta W37A, alpha Y140G, and alpha Y140A, hinge region variants causing large kinetic changes, and for beta Y145G. Such hybrids permit measurements of the kinetics of CO binding to only the heme-containing alpha or beta subunits within the unliganded hemoglobin tetramer. Mutations at beta 37 and alpha 140 have global effects on the T state, increasing the rates of CO binding to both types of subunits. Mutation of beta Y145 has a large effect on the beta subunits in the deoxygenated T state, but very little effect on the alpha subunits. Oxygen equilibria measurements on the crystalline T state of beta W37E also indicate large affinity increases in both subunits of this variant. The overall oxygen equilibria of the variant hemoglobins in solution are sensitive to numerous variables besides the properties of the deoxygenated T state. In contrast to CO combination kinetics, the residues whose alterations cause the largest changes in overall oxygen equilibria in solution are scattered seemingly randomly within the alpha 1 beta 2 interface.     

The beta1 cytoplasmic domain regulates the laminin-binding specificity of the alpha7X1 integrin

During muscle development, the laminin-specific alpha7 integrin is alternatively spliced in the putative ligand-binding domain to yield either the alpha7X1 or the alpha7X2 variant. The relative level of alpha7X1 and alpha7X2 is developmentally regulated. Similarly, the partner beta1 integrin cytoplasmic domain is converted from the beta1A to the beta1D splice variant. To determine whether beta1D modulates the activity of the alpha7 receptor, cells were transfected with alpha7X1 and beta1D cDNA. alpha7X1 coupled with beta1A failed to adhere to laminin-1, whereas cotransfectants expressing alpha7X1 and beta1D showed strong adhesion. Interestingly, alpha7X1 complexed with beta1A and beta1D displayed the same level of poor adhesion to laminin-2/4 or strong adhesion to laminin-10/11. These findings indicate that alpha7 function is regulated not only by X1/X2 in its extracellular domain but also by beta1 cytoplasmic splice variants. It is likely that expression of beta1D alters alpha7X1 binding to laminin isoforms by a process related to ligand affinity modulation. Functional regulation of alpha7beta1 by developmentally regulated splicing events may be important during myogenic differentiation and repair because the integrin mediates adhesion, motility, and cell survival.     

The use of naturally occurring hybrid variants of chloramphenicol acetyltransferase to investigate subunit contacts.

1. Hybrids of the tetrameric enzyme chloramphenicol acetyltransferase (EC 2.3.1.28) were formed in vivo in a strain of Escherichia coli which harbours two different plasmids, each of which normally confers chloramphenicol resistance and specifies an easily distinguished enzyme variant (type I or type III) which is composed of identical subunits. Cell-free extracts of the dual-plasmid strain were found to contain five species of active enzyme, two of which were the homomeric enzymes corresponding to the naturally occurring tetramers of the type-I (beta 4) and type-III (alpha 4) enzymes. The other three variants were judged to be the heteromeric hybrid variants (alpha 3 beta, alpha 2 beta 2, alpha beta 3). 2. The alpha 3 beta and alpha 2 beta 2 hybrids of chloramphenicol acetyltransferase were purified to homogeneity by combining the techniques of affinity and ion-exchange chromatography. The alpha beta 3 variant was not recovered and may be unstable in vitro. 3. The unique lysine residues that could not be modified with methyl acetimidate in each of the native homomeric enzymes were also investigated in the heteromeric tetramers. 4. Lysine-136 remains buried in each beta subunit of the parental (type I) enzyme and in each of the hybrid tetramers. Lysine-38 of each alpha subunit is similarly unreactive in the native type-III chloramphenicol acetyltransferase (alpha 4), but in the alpha 2 beta 2 hybird lysine-38 of each alpha subunit is fully exposed to solvent. Another lysine residue, fully reactive in the alpha 4 enzyme, was observed to be inaccessible to modification in the symmetrical hybrid. The results obtained for the alpha 3 beta enzyme suggest that lysine-38 in two subunits and a different lysine group (that identified in the alpha 2 beta 2 enzyme) in the third alpha subunit are buried. 5. A tentative model for the subunit interactions of chloramphenicol acetyltransferase is proposed on the basis of the results described.     

A carbohydrate structural variant of MM glycoprotein (glycophorin A)

A variant of the MM glycoprotein (glycophorin A) was isolated from erythrocyte membranes of two individual donors, a mother (L.G.) and daughter (V.W.). This glycoprotein was found to be a carbohydrate variant in which, for both donors, certain O-glycosidically linked saccharides retained the core structure consisting of NeuAc(alpha 2,3)Gal(beta 1,3)GalNAc that is common to all O-linked saccharides of the MN glycoproteins, and, in addition, contained substituents, of varying chain lengths, on the primary carbinol of GalNAc. These saccharides were released from the polypeptide by beta-elimination in the presence of sodium borohydride, and aspects of their structure were investigated by glycosidase digestion and periodate oxidation. Thus, the smallest variant structure was deduced to be NeuAc(alpha 2,3)Gal(beta 1,3)[GlcNAc(beta 1,6)]H2GalNAc. The 6-O-linked GlcNAc appears to serve as the focus of further chain elongation reactions, involving alternate additions of Gal and GlcNAc residues and leading to the formation of several homologous structures. Two such structures, NeuAc(alpha 2,3)Gal(beta 1,3)[GlcNAc(beta 1,?) Gal(beta 1,3/4)GlcNAc(beta 1,6)]H2GalNAc and NeuAc(alpha 2,3) Gal(beta 1,3)[Gal(beta 1,3/4)GlcNAc(beta 1,6)]H2GalNAc were the predominant species present. A larger saccharide was also isolated and its partial sequence was determined to be Gal(beta 1,3/4)GlcNAc(beta 1,?)[Gal(beta 1,3/4)Glc-NAc(beta 1,?)] Gal(beta 1,3/4)GlcNAc(beta 1,6)[NeuAc(alpha 2,3)Gal-(beta 1,3)]H2GalNAc. Because the peptide portion of these glycoproteins contains two methionine residues, it was possible to isolate two CNBr glycopeptides from separate regions of the molecule, and to assess the distribution of these variant structures in the polypeptide. The saccharides were linked to about 2-3 Ser and/or Thr residues in the donor LG glycoprotein and one of the attachment sites was located within the CNBr glycooctapeptide representing the NH2 terminus. Considerable heterogeneity in saccharide structure was documented for this site, and it is likely that such heterogeneity occurs also at other sites. The variant saccharides bear structural similarities to the core region of O-linked saccharides of certain blood group-active mucins and ovarian cyst secretions, and to the outer sequences of N-linked carbohydrate units (I-, i-active) of the major glycoprotein of human erythrocytes, band 3. The structures of the variant saccharides suggest that they may be potential precursors of H blood group-active carbohydrates, present in varying degrees of maturity, and attached to an integral protein of erythrocytes.     

Structural analysis of lipooligosaccharide produced by Neisseria gonorrhoeae, strain MS11mk (variant A): a precursor for a gonococcal lipooligosaccharide associated with virulence.

We studied the structure of the lipooligosaccharide (LOS) that is produced by a variant A of strain MS11mk. This variant produces a single LOS that is recognized by monoclonal antibody (MAb) 2-1-L8. In a recent study of the pathogenesis of Neisseria gonorrhoeae in male volunteers, variant A gave rise to other phase variants that produce higher molecular weight LOSs, and these LOS were associated with virulence. Definition of the structure of the variant A LOS is important to understand the biosynthesis of LOS and its expression in vivo. The dephosphorylated oligosaccharide (OS) structure derived from the variant A LOS was analyzed by two-dimensional NMR and methylation analysis. The OS structure was found to be a truncated form of the LOS produced by strain F62 [Yamasaki et al. (1991) Biochemistry 30, 10566-10575]; the variant A OS is a hexamer, a beta-lactosyl residue linked to a tetrasaccharide: Gal beta 1-->4Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->KDO. We determined that the variant A LOS is a precursor for the synthesis of higher MW LOS. We also studied expression of the MAb 2-1-L8-defined epitope present on the variant A LOS. Our data indicate that the MAb-defined epitope is not a linear beta-lactosyl residue but its specificity is directed toward the phosphorylated GlcNAc-Hep-Hep residue. Since this MAb binds to gonococci, at least part of the phosphorylated diheptose area is exposed on the gonococcal surface.     

A proton nuclear magnetic resonance study of the quaternary structure of human homoglobins in water.

Proton nuclear magnetic resonance spectra of human hemoglobins in water reveal several exchangeable protons which are indicators of the quaternary structures of both the liganded and unliganded molecules. A comparison of the spectra of normal human adult hemoglobin with those of mutant hemoglobins Chesapeake (FG4alpha92 Arg yields Leu), Titusville (G1alpha94 Asp yields Asn), M Milwaukee (E11beta67 Val yields Glu), Malmo (FG4beta97 His yields Gln), Kempsey (G1beta99 Asp yields Asn), Yakima (G1beta99 Asp yields His), and New York (G15beta113 Val yields Glu), as well as with those of chemically modified hemoglobins Des-Arg(alpha141), Des-His(beta146), NES (on Cys-beta93)-Des-Arg(alpha141), and spin-labeled hemoglobin [Cys-beta93 reacted with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide], suggests that the proton in the important hydrogen bond between the tyrosine at C7alpha42 and the aspartic acid at G1beta99, which anchors the alpha1beta2 subunits of deoxyhemoglobin (a characteristic feature of the deoxy quaternary structure), is responsible for the resonance at -9.4 ppm from water at 27 degrees. Another exchangeable proton resonance which occurs at -6.4 ppm from H2O is a spectroscopic indicator of the deoxy structure. A resonance at -5.8 ppm from H2O, which is an indicator of the oxy conformation, is believed to originate from the hydrogen bond between the aspartic acid at G1alpha94 and the asparagine at G4beta102 in the alpha1beta2 subunit interface (a characteristic feature of the oxy quaternary structure). In the spectrum of methemoglobin at pH 6.2 both the -6.4- and the -5.8ppm resonances are present but not the -9.4-ppm resonance. Upon the addition of inositol hexaphosphate to methemoglobin at pH 6.2, the usual resonance at -9.4 ppm is shifted to -10 ppm and the resonance at 6.4 ppm is not observed. In the spectrum of methemoglobin at pH greater than or equal to 7.6 with or without inositol hexaphosphate, the resonance at -5.8 ppm is present, but not those at -10 and -6.4 ppm, suggesting that methemoglobin at high pH has an oxy-like structure. Two resonances (at -8.2 and -7.3 ppm) which remain invariant in the two quaternary structures could come from exchangeable protons in the alpha1beta1 subunit interface and/or other exchangeable protons in the hemoglobin molecule which undergo no conformational changes during the oxygenation process. These exchangeable proton resonances serve as excellent spectroscopic probes of the quaternary structures of the subunit interfaces in studies of the molecular mechanism of cooperative ligand binding to hemoglobin.     

Distinct and overlapping ligand specificities of the alpha 3A beta 1 and alpha 6A beta 1 integrins: recognition of laminin isoforms.

The ligand specificity of the alpha 3A beta 1 integrin was analyzed using K562 cells transfected with full-length alpha 3A cDNA and was compared with that of alpha 6A beta 1 in similarly transfected K562 cells. Clones were obtained that showed comparable surface expression of either alpha 3A beta 1 or alpha 6A beta 1 integrins. Those expressing alpha 3A beta 1 attached to and spread on immunopurified human kalinin and cellular matrices containing human kalinin, which is a particular isoform of laminin. In addition, alpha 3A transfectants adhered to bovine kidney laminins possessing a novel A chain variant. Binding to kalinin was blocked by a monoclonal antibody against the A chain constituent of kalinin and adhesion to both kalinin and kidney laminins by anti-alpha 3 and beta 1 monoclonal antibodies. The alpha 3A transfected cells bound more strongly to kalinin and bovine kidney laminins after treatment with the beta 1 stimulatory antibody TS2/16. A distinctly weaker and activation-dependent adhesion of alpha 3A transfectants was observed on human placental laminins possessing the Am chain variant (merosin), and no adhesion occurred on bovine heart laminins and murine EHS tumor laminin. Further inactive substrates were fibronectin, nidogen, and collagen types IV and VI, indicating that the alpha 3A beta 1 integrin is a much less promiscuous receptor than thought before. By contrast, alpha 6A transfected cells adhered to all laminin isoforms when stimulated with TS2/16. Adhesion also occurred only on bovine kidney laminins in the absence of TS2/16. These results demonstrate that both alpha 3A beta 1 and alpha 6A beta 1 integrins are typical laminin receptors but that their affinity and activation dependence for binding to various laminin isoforms differ considerably.     

Subunit composition of G(o) proteins functionally coupling galanin receptors to voltage-gated calcium channels.

The neuropeptide galanin is widely expressed in the central nervous system and other tissues and induces different cellular reactions, e.g. hormone release from pituitary and inhibition of insulin release from pancreatic B cells. By microinjection of antisense oligonucleotides we studied the question as to which G proteins mediate the galanin-induced inhibition of voltage-gated Ca2+ channels in the rat pancreatic B-cell line RINm5F and in the rat pituitary cell line GH3. Injection of antisense oligonucleotides directed against alpha 01, beta 2, beta 3, gamma 2 and gamma 4 G protein subunits reduced the inhibition of Ca2+ channel current which was induced by galanin, whereas no change was seen after injection of cells with antisense oligonucleotides directed against alpha i, alpha q, alpha 11, alpha 14, alpha 15, beta 1, beta 4, gamma 1, gamma 3, gamma 5, or gamma 7 G protein subunits or with sense control oligonucleotides. In view of these data and of previous results, we conclude that the galanin receptors in GH3 and in RINm5F cells couple mainly to the G(0) protein consisting of alpha 01 beta 2 gamma 2 to inhibit Ca2+ channels and use alpha 01beta 3 gamma 4 less efficiently. The latter G protein composition was previously shown to be used by muscarinic M4 receptors to inhibit Ca2+ channels.     

Urinary oligosaccharides of GM1-gangliosidosis. Different excretion patterns of oligosaccharides in the urine of type 1 and type 2 subgroups

Oligosaccharide patterns obtained by gel filtration of the urine of GM1-gangliosidosis Type 1 patients are quite different from those of GM1-gangliosidosis Type 2. By studies of oligosaccharides in the four major peaks obtained from the Type 1 subgroup using sequential exoglycosidase digestion, methylation analysis, and periodate oxidation, the structures of 15 oligosaccharides: Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6Man beta 1 leads to 4GlcNAc, Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6(Gal beta 1 leads to 4Glc NAc beta 1 leads to 2Man alpha 1 leads to 3)Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 6(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 6[Gal beta 1 leads to 4GlcNAc beta 1 leads to 4(Gal beta 1 leads to 4GlcNAc beta 1 leads to 2)Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc, Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 6, and 3(Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3 and 6)Man beta 1 leads to 4GlcNAc, (formula see text) were elucidated. The amounts of total oligosaccharides excreted in the urine of the Type 2 subgroup were approximately one-tenth of those of Type 1. Moreover, the last eight oligosaccharides shown above, which have a Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to outer chain, were completely missing in the urine of Type 2.     

Hemoglobin New Mexico: beta 100 (G2) Pro----Arg. A variant hemoglobin associated with erythrocytosis

Hemoglobin New Mexico beta 100 Pro----Arg was found in a 4-year-old black male and represents a new mutation. The propositus is also heterozygous for Hb S. The variant shows high oxygen affinity, reduced cooperatively, and a lowered alkaline Bohr effect. Addition of allosteric effectors leads to improved cooperativity and a Bohr effect that is similar to that of Hb A. The high percentage of the variant (53.5%) and its increased oxygen affinity result in erythrocytosis in this patient. The hemoglobin level and packed cell volume values are elevated. In spite of these factors the patient appears healthy and shows no discomfort. The altered oxygen-linked properties of this variant can be related to the fact that the substituted residue contributes to the alpha 2 beta 1/alpha 1 beta 2 subunit interface, an area that is critical not only to the allosteric transitions between the oxy and deoxy states but also to stabilizing the hemoglobin tetrameer.     

Beta-adrenoceptor control of G protein function in the neonate: determinant of desensitization or sensitization

Neonatal beta-adrenoceptors (beta-ARs) are resistant to agonist-induced desensitization. We examined the functioning of G(i) and G(s) after repeated administration of beta-AR agonists to newborn rats. Isoproterenol (beta(1)/beta(2) agonist) obtunded G(i) function in the heart but not the liver; in contrast, terbutaline, a beta(2)-selective agonist, enhanced G(i) function. Isoproterenol, but not terbutaline, increased membrane-associated G((s)alpha), which would enhance receptor function. In addition, isoproterenol increased and terbutaline maintained the proportion of the short-splice (S) variant of G((s)alpha) in the membrane fraction; G((s)alpha)S is functionally more active than the long-splice variant. Either isoproterenol or terbutaline treatment increased G((s)alpha) in the cytosolic fraction, a characteristic usually associated with desensitization in the adult. Decreased G(i) activity, coupled with increased membrane-associated G((s)alpha) concentrations and maintenance or increases in membrane G((s)alpha)S, provide strong evidence that unique effects on G protein function underlie the ability of the immature organism to sustain beta-AR cell signaling in the face of excessive or prolonged stimulation; these mechanisms also contribute to tissue selectivity of the effects of beta-agonists with divergent potencies toward different beta-AR subtypes.     

Cell surface sialylation and tumor metastasis. Metastatic potential of B16 melanoma variants correlates with their relative numbers of specific penultimate oligosaccharide structures

Numerous investigations suggest that cell surface glycoconjugates, and in particular sialic acids, are directly involved in determining the metastatic phenotype. To further evaluate this hypothesis, we have used a variety of techniques to probe the cell surfaces of several metastatic variants of the murine B16 melanoma that were selected for experimental lung-colonizing ability (Fidler, I. (1973) Nature 242, 148-149) or for their ability to spontaneously metastasize from the site of a subcutaneous injection (Stackpole, C. W., Alterman, A. L., and Fornabaio, D. M. (1985) Invasion & Metastasis 5, 125-142). Using a highly sensitive high performance liquid chromatography sialic acid assay in conjunction with Vibrio cholerae sialidase, we find that none of these metastatic variants differ significantly in their overall levels of cell surface sialic acid. Using highly purified, linkage-specific sialyltransferases, in conjunction with specific glycosidases, to probe the cell surface saccharide topography of specific penultimate oligosaccharides, we also find no significant differences between the efficient lung-colonizing variant, B16-F10 and the poorly-colonizing B16-F1 or B16-Flr variants. In contrast, the spontaneously metastatic variants examined contain substantially different levels of specific penultimate sialylation sites. The tumorigenic but nonmetastatic B16-LM3/G3.26 variant contains 4-fold more penultimate Gal beta 1-3GalNAc sialylation sites than the tumorigenic and highly metastatic B16-LM3/G3.12 variant when CMP[3H]NeuAc and the alpha 2-3Gal beta 1-3GalNAc sialyltransferase are used to probe the melanoma cell surfaces. Several prominent glycoconjugates of apparent Mr 43,000, 40,000, and 30,000 are especially evident upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the nonmetastatic cells. The nonmetastatic variant also contains 2-fold more Gal beta 1-4GlcNAc sialylation sites than the metastatic variant when the alpha 2-6Gal beta 1-4GlcNAc sialyltransferase is used as a cell surface probe. In this case, glycoconjugates of apparent Mr 74,000, 45,000, and 43,000 are more prominently observed on the cell surfaces of the nonmetastatic variant. These data indicate that the differences in lung-colonizing abilities of B16 melanoma metastatic variants do not correlate with the numbers or sialylation states of specific penultimate oligosaccharide structures on their surfaces. However, the relative levels of specific penultimate saccharide structures do correlate with the ability of the cells to undergo spontaneous metastasis from a subcutaneous tumor.     

G Protein beta subunit types differentially interact with a muscarinic receptor but not adenylyl cyclase type II or phospholipase C-beta 2/3

In comparison with the alpha subunit of G proteins, the role of the beta subunit in signaling is less well understood. During the regulation of effectors by the betagamma complex, it is known that the beta subunit contacts effectors directly, whereas the role of the beta subunit is undefined in receptor-G protein interaction. Among the five G protein beta subunits known, the beta(4) subunit type is the least studied. We compared the ability of betagamma complexes containing beta(4) and the well characterized beta(1) to stimulate three different effectors: phospholipase C-beta2, phospholipase C-beta3, and adenylyl cyclase type II. beta(4)gamma(2) and beta(1)gamma(2) activated all three of these effectors with equal efficacy. However, nucleotide exchange in a G protein constituting alpha(o)beta(4)gamma(2) was stimulated significantly more by the M2 muscarinic receptor compared with alpha(o)beta(1)gamma(2). Because alpha(o) forms heterotrimers with beta(4)gamma(2) and beta(1)gamma(2) equally well, these results show that the beta subunit type plays a direct role in the receptor activation of a G protein.     

Members of the Gq alpha subunit gene family activate phospholipase C beta isozymes.

The relative specificities of members of the G alpha q family of GTP-binding proteins were tested for their ability to activate different phosphoinositide-specific phospholipase C (PI-PLC) beta isozymes. Cos-7 cells were transfected with cDNA corresponding to G alpha q, G alpha 11, G alpha 14, and G alpha 16. Most of the recombinant protein was bound to the cell membrane and these membranes were washed to elute endogenous PI-PLC activity. The membrane preparation was reconstituted with purified preparations of the PI-PLC beta isozymes and guanosine 5'-O-thiotriphosphate (GTP gamma S)-stimulated enzyme activity was measured. All four proteins of the G alpha q family were found to stimulate PI-PLC beta 1, with G alpha q and G alpha 11 being most efficient. On the other hand, G alpha 16 was found to most effectively activate PI-PLC beta 2, while G alpha q, G alpha 11, and G alpha 14 showed less stimulation. Specific anti- G alpha 16 antibody blocked the stimulation of both PI-PLC beta 1 and PI-PLC beta 2 in the enriched membrane fraction. We conclude that there is specificity in the interaction of different members of the Gq family with different PI-PLC beta effectors. This specificity may be important in generating tissue- or receptor-specific responses in vivo.     

Integrin alpha 6 beta 4 forms a complex with the cytoskeletal protein HD1 and induces its redistribution in transfected COS-7 cells.

The integrin alpha 6 beta 4 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its presence in these structures is dependent on the beta 4 cytoplasmic domain but it is not known whether beta 4 interacts directly with keratin filaments or by interaction with other proteins. In this study, we have investigated the interaction of GST-cyto beta 4A fusion proteins with cellular proteins and demonstrate that a fragment of beta 4A, consisting of the two pairs of fibronectin type III repeats, separated by the connecting segment, forms a specific complex containing a 500-kDa protein that comigrates with HD1, a hemidesmosomal plaque protein. A similar protein was also bound by a glutathione S-transferase fusion protein containing the cytoplasmic domain of a variant beta 4 subunit (beta 4B), in which a stretch of 53 amino acids is inserted in the connecting segment. Subsequent immunoblot analysis revealed that the 500-kDa protein is in fact HD1. In COS-7 cells, which do not express alpha 6 beta 4 or the hemidesmosomal components BP230 and BP180, HD1 is associated with the cytoskeleton, but after transfecting the cells with cDNAs for human alpha 6 and beta 4, it was, instead, colocalized with alpha 6 beta 4 at the basal side of the cells. The organization of the vimentin, keratin, actin, and tubulin cytoskeletal networks was not affected by the expression of alpha 6 beta 4 in COS-7 cells. The localization of HD1 at the basal side of the cells depends on the same region of beta 4 that forms a complex containing HD1 in vitro, since the expression of alpha 6 with a mutant beta 4 subunit that lacks the four fibronectin type III repeats and the connecting segment did not alter the distribution of HD1. The results indicate that for association of alpha 6 beta 4 with HD1, the cytoplasmic domain of beta 4 is essential. We suggest that this association may be crucial for hemidesmosome assembly.     

Genetic identification of residues involved in association of alpha and beta G-protein subunits.

The GPA1, STE4, and STE18 genes of Saccharomyces cerevisiae encode the alpha, beta, and gamma subunits, respectively, of a G protein involved in the mating response pathway. We have found that mutations G124D, W136G, W136R, and delta L138 and double mutations W136R L138F and W136G S151C of the Ste4 protein cause constitutive activation of the signaling pathway. The W136R L138F and W136G S151C mutant Ste4 proteins were tested in the two-hybrid protein association assay and found to be defective in association with the Gpa1 protein. A mutation at position E307 of the Gpa1 protein both suppresses the constitutive signaling phenotype of some mutant Ste4 proteins and allows the mutant alpha subunit to physically associate with a specific mutant G beta subunit. The mutation in the Gpa1 protein is adjacent to the hinge, or switch, region that is required for the conformational change which triggers subunit dissociation, but the mutation does not affect the interaction of the alpha subunit with the wild-type beta subunit. Yeast cells constructed to contain only the mutant alpha and beta subunits mate and respond to pheromones, although they exhibit partial induction of the pheromone response pathway. Because the ability of the modified G alpha subunit to suppress the Ste4 mutations is allele specific, it is likely that the residues defined by this analysis play a direct role in G-protein subunit association.     

Activation of phospholipase C by alpha 1-adrenergic receptors is mediated by the alpha subunits of Gq family.

High efficiency transient transfection of Cos-7 cells was previously used to establish the functional coupling between G alpha q/G alpha 11 and phospholipase C beta 1 (Wu, D., Lee, C-H., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 1811-1817). Here the same system was used to study the functional coupling between other guanine nucleotide-binding regulatory protein (G-protein) alpha subunits and phospholipases and to study which G alpha subunits mediate the activation of phospholipase C by the alpha 1-adrenergic receptor subtypes, alpha 1 A, alpha 1 B, and alpha 1 C. We found that G alpha 14 and G alpha 16 behaved like G alpha 11 or G alpha q, i.e. they could activate endogenous phospholipases in Cos-7 cells in the presence of AIFn. The synergistic increase in inositol phosphate release in Cos-7 cells after they were cotransfected with cDNAs encoding G alpha subunits and phospholipase C beta 1 indicates that both G alpha 16 and G alpha 14 can activate phospholipase C beta 1. The activation of phospholipase C beta 1 was restricted to members of the Gq subfamily of alpha subunits. They activated phospholipase C beta 1 but not phospholipase C gamma 1, gamma 2, or phospholipase C delta 3. The cotransfection of Cos-7 cells with cDNAs encoding three different alpha 1-adrenergic receptors and G alpha q or G alpha 11 leads to an increase in norepinephrine-dependent inositol phosphate release. This indicates that G alpha q or G alpha 11 can mediate the activation of phospholipase C by all three subtypes of alpha 1-adrenergic receptors. With the same assay system, G alpha 16 and G alpha 14 appear to be differentially involved in the activation of phospholipase C by the alpha 1-adrenergic receptors. The alpha 1 B subtype receptor gave a ligand-mediated synergistic response in the cells cotransfected with either G alpha 14 or G alpha 16. However, the alpha 1 C receptor responded in cells cotransfected with G alpha 14 but not G alpha 16, and the alpha 1 A receptor showed little synergistic response in cells transfected with either G alpha 14 or G alpha 16. The ability of the alpha 1 A and alpha 1 C receptors to activate phospholipase C through G alpha q and G alpha 11 was also demonstrated in a cell-free system.(ABSTRACT TRUNCATED AT 400 WORDS)