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N Gautam

Publications and source records attributed to N Gautam.

49 records · Page 3Linked to original sources
PubMed1995

Receptor-G protein coupling is established by a potential conformational switch in the beta gamma complex.

Receptor-G protein interaction is characterized by cycles of association and dissociation. We present evidence which indicates that during receptor-G protein interaction, the C-terminal tail of the G protein gamma subunit, which is masked in the beta gamma complex, is exposed and establishes high-affinity contact with the receptor. This potential conformational switch provides a mechanism to regulate receptor-G protein coupling. This switch may also be significant for the role of the beta gamma complex in regulation of effector function.

Amino Acid Sequence↗
PubMed1994

A farnesylated domain in the G protein gamma subunit is a specific determinant of receptor coupling.

The interaction between receptor and a heterotrimeric G protein (alpha beta gamma) is thought to involve the intracellular loops of receptors and specific domains on the G protein. Here we show that a chemically farnesylated peptide (P5far) specific to the carboxyl-terminal domain (amino acids 60-71: DKNPFKELKGGC) of the gamma subunit of the G protein, Gt, directly stabilizes the active form of rhodopsin, metarhodopsin II (M II), and also uncouples rhodopsin-Gt interaction. Peptide activity is significantly affected by the absence of the isoprenoid moiety. Moreover, we show that altering the amino acid sequence of the farnesylated peptide by randomizing the sequence, substituting hydrophobic with hydrophilic residues (F64T; L67S) or deleting amino acids 60-66 significantly reduces the ability of the peptide to stabilize M II. This indicates that both the farnesyl moiety and the structure of the gamma subunit tail are specific determinants of receptor-G protein interaction. These results also suggest a general function for the family of G protein gamma subunits in signaling.

Amino Acid Sequence↗
PubMed1993

Specific interaction with rhodopsin is dependent on the gamma subunit type in a G protein.

Interaction with a receptor is the first step in the process of signal transduction by heterotrimeric (alpha beta gamma) G proteins. We have examined the role of the G protein gamma subunit in interaction between a receptor (rhodopsin) and a G protein, transducin (Gt). We have obtained recombinant beta gamma complexes containing the same beta subunit but three different gamma subunit types by expressing them in the baculovirus/insect cell system. We show that the different recombinant beta gamma complexes interact equally well with the alpha subunit of transducin (alpha t) but only the gamma subunit specific to rod photoreceptors (gamma 1) is able to support interaction of alpha t with rhodopsin. This indicates a direct role for the G protein gamma subunits, which are a family of proteins with diverse structures, in conferring specificity to receptor-G protein interaction.

Amino Acid Sequence↗
PubMed1993

Proper processing of a G protein gamma subunit depends on complex formation with a beta subunit.

G protein beta and gamma subunits function as a tightly associated complex. We show that complex formation with the beta subunit is a critical step for post-translational processing of a gamma subunit. When expressed alone in a cell line, the gamma 3 subunit type is isoprenylated but degraded; co-expression with the beta 1 subunit type stabilizes the gamma 3 protein. Furthermore, our experiments with partial cell fractionation indicate that the gamma 3 protein is localized differently in the cell depending on whether or not it is bound to the beta subunit. Binding of the gamma subunit to the beta subunit is thus one of the prerequisites for the appropriate intracellular localization of the beta gamma complex and potentially, for normal G-protein function.

Animals↗
PubMed1992

Interaction between G-protein beta and gamma subunit types is selective.

Signal-transducing guanine nucleotide-binding proteins (G proteins) are made up of three subunits, alpha, beta, and gamma. Each of these subunits comprises a family of proteins. The rules for association between members of one family with members of another to form a multimer are not known; it is not clear whether associations are specific or nonspecific. Other than transducin (Gt), the G protein in rod photoreceptors, most purified G proteins contain more than one subtype of beta or gamma subunits. The Gt alpha subunit is associated only with beta 1 and gamma 1. It is not known whether this specificity is due to the differential expression of these subunit types in a cell type or due to intrinsically different affinities between different beta and gamma subunit types. We have used a transfected cell assay system to examine the association of the beta 1, beta 2, and beta 3 proteins with the gamma 1 and gamma 2 proteins. Results show that gamma 1 does not associate with beta 2 and that beta 3 does not associate with gamma 1 or gamma 2. Differences in affinities between types of G protein subunits will impose restrictions on the formation of certain heterotrimers and determine which G protein will be active in a cell. A chimeric molecule of beta 1 and beta 2 was used to broadly map the regions on these subunits that determine specificity of association.

Animals↗
PubMed1991

Diversity of G proteins in signal transduction.

The heterotrimeric guanine nucleotide-binding proteins (G proteins) act as switches that regulate information processing circuits connecting cell surface receptors to a variety of effectors. The G proteins are present in all eukaryotic cells, and they control metabolic, humoral, neural, and developmental functions. More than a hundred different kinds of receptors and many different effectors have been described. The G proteins that coordinate receptor-effector activity are derived from a large gene family. At present, the family is known to contain at least sixteen different genes that encode the alpha subunit of the heterotrimer, four that encode beta subunits, and multiple genes encoding gamma subunits. Specific transient interactions between these components generate the pathways that modulate cellular responses to complex chemical signals.

Amino Acid Sequence↗
PubMed1991

G-protein beta gamma dimers. Membrane targeting requires subunit coexpression and intact gamma C-A-A-X domain.

Attachment of heterotrimeric G-proteins to the inner face of the plasma membrane is fundamental to their role as signal transducers by allowing interaction with both receptors and effectors. Certain G-protein alpha subunits are anchored to the membrane by covalent myristoylation. The beta gamma complex is required for G-protein interaction with receptors and is independently membrane associated through an unknown mechanism. A series of carboxyl-terminal modifications including isoprenylation which may contribute to membrane attachment has been identified recently in G-protein gamma subunits. Expression and membrane targeting of beta and gamma subunits were examined in COS cells. The expression of either subunit was found to require cotransfection with both beta and gamma cDNAs. Mutation of the carboxyl-terminal cysteine residue of gamma shown to undergo isoprenylation and carboxymethyl-esterification preserved beta gamma expression but blocked isoprenylation and membrane attachment. These results implicate the carboxyl-terminal processing of G-protein gamma subunits and beta coexpression as necessary and sufficient for membrane targeting of the beta gamma complex.

Blotting, Northern↗
PubMed1990

G protein diversity is increased by associations with a variety of gamma subunits.

Guanine nucleotide-binding regulatory proteins (G proteins) are heterotrimeric proteins that transduce extracellular signals into intracellular changes. Functionally different G proteins have been identified by their different alpha subunits. The beta and gamma subunits have been assumed to constitute a common pool shared among various G protein heterotrimers. Two gamma subunits (gamma 3 and gamma 4) have been identified through molecular cloning; these are in addition to two subunits (gamma 1 and gamma 2) that were previously characterized. G proteins purified from a variety of mammalian tissues were examined with antisera specific to three of the gamma subunits. The antisera react with different gamma subunits associated with some of the purified G proteins but not all. This demonstrates that different G protein heterotrimers from different tissues carry structurally distinct members of the gamma-subunit family. Diversity in the structure of the gamma as well as the alpha and beta subunits and preferential associations between members of subunit families increase structural and possibly functional diversity of G proteins.

Amino Acid Sequence↗
PubMed1989

A G protein gamma subunit shares homology with ras proteins.

Guanine nucleotide binding proteins (G proteins) that transduce signals from cell surface receptors to effector molecules are made up of three subunits, alpha, beta, and gamma. A complementary DNA clone that encodes a 71-amino acid protein was isolated from bovine brain; this protein contains peptide sequences that were derived from the purified gamma subunit of Gi and Go. The primary sequence of this G protein gamma subunit (G gamma) has 55 percent homology to the gamma subunit of transducin (T gamma) and also has homology to functional domains of mammalian ras proteins. The probe for isolating the clone was generated with the use of the polymerase chain reaction (PCR). The extent of divergence between T gamma and G gamma, the isolation of homologous PCR-generated fragments, and the differences between the predicted amino acid sequence of G gamma and that derived from the gamma subunit of Gi and Go indicate that gamma subunits are encoded by a family of genes.

Amino Acid Sequence↗
PubMed1988

Mutated forms of phosphoglycerate mutase in yeast affect reversal of metabolic flux. Effect of reversible and irreversible function of an enzyme on pathway reversal.

Phosphoglycerate mutase (GPM) functions reversibly in the glycolytic pathway. Mutations altering the reversibility of GPM have been obtained in the yeast, Saccharomyces cerevisiae. While wild-type cells grow on glycolytic (glucose) or gluconeogenic (ethanol) substrates, cells with altered GPMs fall into three categories based on their phenotypes 1) glucose- ethanol-, 2) glucose+ ethanol-, and 3) glucose- ethanol+. Cells with the first two phenotypes possessed GPMs that functioned irreversibly in the glycolytic direction. Cells that were glucose- ethanol+ possessed an enzyme that functioned reversibly. All of the altered GPMs had maximal velocities that were less than 3% of the wild-type level. The properties of the altered GPMs studied here provide a rationale for the occurrence in the glycolytic pathway of several glycolytic enzymes such as GPM, which function at high velocities in relation to the much smaller metabolic flux that they support. The altered GPMs were purified and estimates of their kinetic constants obtained. Free energy profiles were drawn for catalysis by the wild type and a mutant GPM that functioned irreversibly. The mutant enzyme was very inefficient. It was shown that an enzyme that functions irreversibly at a reaction with a Keq value close to 1 would necessarily be inefficient while it could evolve to be efficient when catalyzing a reaction that has a Keq value much greater than 1. In the glycolytic path this could be the reason for the characteristic presence of enzymes that function irreversibly at reactions with large Keq values.

Algorithms↗
PubMed1988

The 35- and 36-kDa beta subunits of GTP-binding regulatory proteins are products of separate genes.

The wide range of functions attributed to GTP-binding regulatory proteins (G proteins) is reflected in the structural diversity which exists among the alpha, beta, and gamma subunits of G proteins. Recently two cDNA clones encoding beta subunits, beta 1 and beta 2, were isolated from bovine and human cDNA libraries. We report here that the beta 2 gene encodes the 35-kilodalton (kDa) component of the beta 35/beta 36 subunit of G proteins and that the beta 1 gene encodes the 36-kilodalton component. The in vitro translation product of the beta 2 cDNA co-migrates with the 35-kDa beta subunit (beta 35), while the in vitro product of the beta 1 cDNA co-migrates with the 36-kDa beta subunit (beta 36) on denaturing polyacrylamide gels. In addition, antisera generated against synthetic beta 2 peptides bind specifically to the beta 35 component of isolated G proteins and to a 35-kDa protein in myeloid cell membranes. Our results suggest that the two beta subunits could serve distinct functions, as they are derived from separate genes which have been highly conserved in evolution.

Amino Acid Sequence↗