Biomedical subjects

N Gautam

Publications and source records attributed to N Gautam.

At least 37 records · Page 2Linked to original sources

PubMed1999

A G protein gamma subunit-specific peptide inhibits muscarinic receptor signaling.

Muscarinic acetylcholine receptors modulate the function of a variety of effectors through heterotrimeric G proteins. A prenylated peptide specific to the G protein gamma5 subunit type inhibits G protein activation by the M2 muscarinic receptor in a reconstitution assay. Scrambling the amino acid sequence of the peptide significantly reduces the efficacy of the peptide. The peptide does not disrupt the G protein heterotrimer. In cultured sympathetic neurons, the gamma5 peptide inhibits modulation of Ca(2+) current by the M4 receptor. Peptide activity is specific, the scrambled peptide and peptides specific to two other members of the G protein gamma subunit family are significantly less effective. The gamma5 peptide has no effect on Ca(2+) current modulation by the alpha2-adrenergic and somatostatin receptors. In addition, the gamma5 peptide inhibits muscarinic receptor signaling in spinal cord slices with specificity. These results support a specific role for G protein gamma subunit types in signal transduction, most likely at the receptor-G protein interface.

Amino Acid Sequence↗

PubMed1999

Chromosomal mapping of five mouse G protein gamma subunits.

Heterotrimeric G proteins, composed of alpha, beta, and gamma subunits, transduce signals from transmembrane receptors to a wide range of intracellular effectors. The G protein gamma subunits, which play an indispensible role in this communication, constitute a large and diverse multigene family. Using an interspecific backcross panel, we have determined the mouse chromosomal locations of five gamma subunit genes: gamma2, gamma8, gamma10, gamma12, and gammaCone. Combined with previous mapping studies, these data indicate that, with the possible exception of gamma1 and gamma11, the G protein gamma subunit genes are well dispersed within the mouse and human genomes.

Animals↗

PubMed1999

KSR-1 binds to G-protein betagamma subunits and inhibits beta gamma-induced mitogen-activated protein kinase activation.

The protein kinase KSR-1 is a recently identified participant in the Ras signaling pathway. The subcellular localization of KSR-1 is variable. In serum-deprived cultured cells, KSR-1 is primarily found in the cytoplasm; in serum-stimulated cells, a significant portion of KSR-1 is found at the plasma membrane. To identify the mechanism that mediates KSR-1 translocation, we performed a yeast two-hybrid screen. Three clones that interacted with KSR-1 were found to encode the full-length gamma10 subunit of heterotrimeric G-proteins. KSR-1 also interacted with gamma2 and gamma3 in a two-hybrid assay. Deletion analysis demonstrated that the isolated CA3 domain of KSR-1, which contains a cysteine-rich zinc finger-like domain, interacted with gamma subunits. Coimmunoprecipitation experiments demonstrated that KSR-1 bound to beta1 gamma3 subunits when all three were transfected into cultured cells. Lysophosphatidic acid treatment of cells induced KSR-1 translocation to the plasma membrane from the cytoplasm that was blocked by administration of pertussis toxin but not by dominant-negative Ras. Finally, transfection of wild-type KSR-1 inhibited beta1 gamma3-induced mitogen-activated protein kinase activation in cultured cells. These results demonstrate that KSR-1 translocation to the plasma membrane is mediated, at least in part, by an interaction with beta gamma and that this interaction may modulate mitogen-activated protein kinase signaling.

Animals↗

PubMed1998

G-protein beta-subunit specificity in the fast membrane-delimited inhibition of Ca2+ channels.

We investigated which subtypes of G-protein beta subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein beta subunits. Gbeta1 and Gbeta2 strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The Gbeta5 subunit produced much weaker effects than Gbeta1 and Gbeta2, whereas Gbeta3 and Gbeta4 were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, Gbeta1 or Gbeta2 coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a alpha1 subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the Gbeta5 subunit interacted weakly, and Gbeta3 and Gbeta4 failed to interact. Together, these results suggest that Gbeta1 and/or Gbeta2 subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel alpha1 subunit is a principal receptor for this inhibition.

Adrenergic Fibers↗

PubMed1998

Structure and mapping of the G protein gamma3 subunit gene and a divergently transcribed novel gene, gng3lg.

The mammalian nervous system is rich in signaling mediated by heterotrimeric (alphabetagamma) G proteins. As an initial step to define the roles that particular gamma subunit types play in signaling, we have begun to clone and characterize those genes that encode gamma subunits enriched within neural tissue. In the present study, we have isolated and characterized the mouse gamma3 subunit gene (Gng3). The gamma3 subunit is expressed abundantly in the brain and at low levels in testes. Gng3 is composed of three exons spanning approximately 1.4 kb. A comparison of Gng3 with the gene structure for five other gamma subtypes indicates that although these proteins are diverse at the amino acid level, their exon-intron boundaries are conserved. Sequence analysis of the 5' flanking region of Gng3 revealed the presence of a novel gene, the gamma3 linked gene (Gng3lg). Gng3 and Gng3lg are organized in a head-to-head fashion with major transcription initiation sites separated by approximately 133 bp. Sequence analysis of a Gng3lg cDNA clone revealed an open reading frame encoding a 410-amino-acid protein of unknown function. Gng3lg transcripts are expressed in a variety of tissues including both brain and testes. Using an interspecific backcross panel, we localized both Gng3 and Gng3lg to the same locus on chromosome 19. The orientation, close proximity, and expression pattern of these two genes raise the distinct possibility that shared regulatory elements are used to control their expression.

Animals↗

PubMed1998

Light-activated rhodopsin induces structural binding motif in G protein alpha subunit.

A large superfamily of transmembrane receptors control cellular responses to diverse extracellular signals by catalyzing activation of specific types of heterotrimeric GTP-binding proteins. How these receptors recognize and promote nucleotide exchange on G protein alpha subunits to initiate signal amplification is unknown. The three-dimensional structure of the transducin (Gt) alpha subunit C-terminal undecapeptide Gtalpha(340-350) IKENLKDCGLF was determined by transferred nuclear Overhauser effect spectroscopy while it was bound to photoexcited rhodopsin. Light activation of rhodopsin causes a dramatic shift from a disordered conformation of Gtalpha(340-350) to a binding motif with a helical turn followed by an open reverse turn centered at Gly-348, a helix-terminating C capping motif of an alphaL type. Docking of the NMR structure to the GDP-bound x-ray structure of Gt reveals that photoexcited rhodopsin promotes the formation of a continuous helix over residues 325-346 terminated by the C-terminal helical cap with a unique cluster of crucial hydrophobic side chains. A molecular mechanism by which activated receptors can control G proteins through reversible conformational changes at the receptor-G protein interface is demonstrated.

Amino Acid Sequence↗

PubMed1998

Structure and chromosomal localization of mouse G protein subunit gamma 4 gene.

The G protein gamma subunits are members of a multigene family and are implicated in determining the specificity of receptor-G protein interaction. The gene structures for many of the gamma subunits remain to be determined. Here, we report the gene structure for the brain-specific gamma 4 subunit and its map position on a mouse chromosome. The gene (Gng4) comprises at least three exons spanning over 20 kb. The 225-bp coding region, which spans two exons, is interrupted by a large 18.2-kb intron whose position is conserved in other gamma subunit genes. There is a putative additional intron in the 5' untranslated region just upstream of the translation initiation codon. Introns are present in most of the other gamma subunits at this position. The mouse Gng4 gene is mapped to chromosome 13.

Amino Acid Sequence↗

PubMed1998

The G-protein betagamma complex.

The vast majority of signalling pathways in mammalian cells are mediated by heterotrimeric (alpha betagamma) G proteins. Reviewed here is regulation of signal transduction by the betagamma complex at different protein interfaces: subunit-subunit, receptor-G protein and G protein-effector. The role of diverse beta and gamma subunit types in achieving specificity in signalling and potentially unidentified functions for these subunits also are discussed.

Amino Acid Sequence↗

PubMed1998

Kinetics of leukocyte-induced changes in endothelial barrier function.

1. Extravasation of polymorphonuclear leukocytes (PMN) and associated plasma leakage are key events in the inflammatory process. The kinetics of PMN-induced changes in endothelial barrier function were studied by means of confluent monolayers of bovine aorta or human umbilical vein endothelial cells (EC), cultured on permeable membranes and mounted in a two-compartment diffusion chamber. The model permitted continuous measurement of transendothelial electrical resistance (TEER), and analysis of protein efflux and PMN migration across the EC monolayer. 2. Transendothelial chemotactic stimulation (fMLP or LTB4) of PMN resting on EC in the upper compartment induced a prompt decline in TEER, followed by an increase in protein flux and transmigration of PMN. Adding the chemoattractant together with PMN in the upper compartment provoked adhesion of PMN, fall in TEER and increase in protein permeability, but no transmigration of PMN, whereas inhibition of PMN adhesion to EC by pretreatment with anti-CD18 mAb prevented all responses to chemotactic stimulation. 3. Chemoattractant-induced adhesion of PMN to the EC monolayer induced a rapid rise in EC cytosolic free Ca2+, similar to that obtained by direct stimulation of EC with histamine, indicating an active response of EC to PMN activation and adhesion. 4. In summary, continuous recording of transendothelial electrical resistance in the in vitro model described permits rapid and sensitive analysis of leukocyte activation-induced effects on EC barrier function. The kinetics and specificity of the EC and PMN responses to chemoattractant stimulation suggest that activated PMN, via adhesion-dependent events, have a direct effect on EC junctional integrity independent of whether transmigration occurs or not.

Animals↗

PubMed1997

G protein betagamma complex-mediated apoptosis by familial Alzheimer's disease mutant of APP.

In familial Alzheimer's disease (FAD), three missense mutations, V642I, V642F and V642G, that co-segregate with the disease phenotype have been discovered in the 695 amino acid form of the amyloid precursor protein APP. Expression of these mutants causes a COS cell NK1 clone to undergo pertussis toxin-sensitive apoptosis in an FAD trait-linked manner by activating the G protein Go, which consists of G alpha(o) and G betagamma subunits. We investigated which subunit was responsible for the induction of apoptosis by V642I APP in NK1 cells. In the same system, expression of mutationally activated G alpha(o) or G alpha(i) induced little apoptosis. Apoptosis by V642I APP was antagonized by the overexpression of the carboxy-terminal amino acids 495-689 of the beta-adrenergic receptor kinase-1, which blocks the specific functions of G betagamma. Co-transfection of G beta2gamma2 cDNAs, but not that of other G beta(x)gamma(z) (x = 1-3; z = 2, 3), induced DNA fragmentation in a manner sensitive to bcl-2. These data implicate G betagamma as a cell death mediator for the FAD-associated mutant of APP.

Alzheimer Disease↗

PubMed1997

Structural determinants for interaction with three different effectors on the G protein beta subunit.

In the yeast two-hybrid system, a 100-residue fragment (beta1A) from the N terminus of the beta1 subunit interacts with domains specific to adenylyl cyclase 2 (AC2), the muscarinic atrial potassium channel (GIRK1), and phospholipase C-beta2 (PLC-beta2). Based on the crystal structure of the G protein, beta1A is composed of an N-terminal alpha helix, a loop, and five beta strands in which the C-terminal four beta strands form a beta sheet, the first of seven sheets that make up the propeller structure of the beta subunit. A mutant of beta1A (L4P, L7P, and L14P), in which the alpha helix was potentially destroyed, interacted poorly with the G protein gamma subunit but effectively with domains of AC2, GIRK1, and PLC-beta2. In contrast, another mutant of beta1A (S72A, D76A, and W82A), in which a network of hydrogen bonds was disrupted, interacted poorly with GIRK1 and PLC-beta2 domains, but effectively with the gamma subunit and the AC2 domain. These results suggest that the proper folding of the first five beta strands in the G protein beta subunit is a requirement for appropriately positioning residues that interact with GIRK1 and PLC-beta2. Furthermore, since mutations that potentially disrupted the folding of these beta strands did not affect interaction with AC2, the structural determinants on the G protein beta subunit for interaction with various effectors may be different.

Adenylyl Cyclases↗

PubMed1997

Tumor cell arrest in the microcirculation: lack of evidence for a leukocyte-like rolling adhesive interaction with vascular endothelium in vivo.

Hematogenous spread of tumor cells and metastasis formation in secondary organs are insidious aspects of cancer. In the present intravital microscopic study in the rabbit mesentery, we examined the in vivo flow behavior of six human tumor cell lines of different histological origin. The tumor cells and human neutrophils were injected locally into a side branch of the superior mesenteric artery upstream of the observed microvascular area in the mesentery. None of the tumor cells behaved similar to the leukocytes of which a substantial fraction rolled along the endothelium of small venules. Thus, the tumor cells passed the same venular segments without interacting with the endothelial lining. Yet, three of the tumor cell lines (HT-29, DLD-1, and HCT-8) were strongly positive for the oligosaccharides Lewis(x), sialyl-Lewis(x), and sialyl-Lewis(a) which are recognized by the endothelial selectins that mediate leukocyte rolling. On the other hand, some tumor cells were trapped in the smallest vessels and remained so throughout the experimental period, apparently due to a discrepancy in size between tumor cells and microvessel lumen. Taken together, our in vivo findings suggest that initial microvascular arrest of metastasizing tumor cells is dependent primarily on mechanical factors rather than on receptor-mediated leukocyte-like adhesive interactions.

Animals↗

PubMed1997

Impaired leucocyte rolling, adhesion and transendothelial migration following cuprophane haemodialysis.

In this study, we investigated how different steps in the extravasation process of leucocytes, i.e. rolling, adhesion and transendothelial migration, are affected by haemodialysis with cuprophane membranes. Human leucocytes obtained from whole blood prior to clinical haemodialysis and from the afferent blood line (post-dialyser) 15 min after the initiation of dialysis were injected into the mesenteric microcirculation of urethane anaesthetized rabbits and analysed for their ability to roll in the microvessels by use of intravital fluorescence microscopy. Moreover, neutrophils from the two leucocyte populations were compared with respect to chemoattractant-induced adhesion and transmigration across confluent monolayers of bovine aortic endothelial cells. Our results show that, as compared with pre-dialysis leucocytes, 15 min of cuprophane haemodialysis impaired leucocyte rolling by 78 +/- 7%, reduced N-formyl-methionyl-leucyl-phenylalanin (fMLP)-induced adhesion by 34 +/- 9%, and abolished transendothelial migration. These findings demonstrate that intradialytical activation of leucocytes during cuprophane haemodialysis severely affects leucocyte functions that are critical in the extravasation process of these cells at inflammatory tissue sites, and thus may help explain the increased susceptibility to infections observed in patients on chronic haemodialysis.

Animals↗

PubMed1996

Bombesin and thrombin affect discrete pools of intracellular calcium through different G-proteins.

In mouse NIH 3T3 cells, the mitogens bombesin and thrombin induced Ca2+ release from intracellular stores. Ca2+ release induced by bombesin was inhibited by the Ca(2+)-ATPase inhibitor thapsigargin, while Ca2+ release induced by thrombin was unaffected by this agent. The Ca(2+)-release response to bombesin was not affected by pertussis toxin, but the response to thrombin was abolished by the toxin. Stable transfectants overexpressing the G-protein subunit type alpha 9 showed an accentuated response to bombesin, indicating that the bombesin receptor was coupled to a Gq-like G-protein. Together, these results show that the two mitogenic receptors are coupled to distinct G-proteins that affect functionally different pools of Ca2+. Organization of signalling pathways in this manner may allow cells to differentially encode information from different signals.

3T3 Cells↗

PubMed1996

A domain on the G protein beta subunit interacts with both adenylyl cyclase 2 and the muscarinic atrial potassium channel.

The G protein betagamma complex modulates the function of a variety of effectors in biological signaling. However, the individual roles of the beta and gamma subunits in this interaction are unknown. Unlike in the case of the alpha subunit, domains on the betagamma complex that contact effectors have not yet been identified. We show here using the yeast two-hybrid system that the beta subunit and not the gamma subunit interacts with domains specific to adenylyl cyclase type 2 (AC2) and the muscarinic receptor-gated atrial inwardly rectifying potassium channel, GIRK1. Different beta subunit types interact with these effector domains with different efficacies. Furthermore, an N-terminal fragment of 100 residues interacts with both these effector domains as effectively as the whole beta subunit. This domain includes the region where the beta subunit contacts with the alpha subunit in the crystal structure and may therefore explain the ability of the alpha subunit to shut off the activity of the betagamma complex.

Adenylyl Cyclases↗

PubMed1996

Differential ability to form the G protein betagamma complex among members of the beta and gamma subunit families.

We have determined the relative abilities of several members of the G protein beta and gamma subunit families to associate with each other using the yeast two-hybrid system. We show first that the mammalian beta1 and gamma3 fusion proteins form a complex in yeast and that formation of the complex activates the reporter gene for beta-galactosidase. Second, the magnitude of reporter activity stimulated by various combinations of beta and gamma subunit types varies widely. Third, the reporter activity evoked by a particular combination of beta and gamma subunit types is not correlated with the expression levels of these subunit types in the yeast cells. Finally, the reporter activity shows a direct relationship with the amount of hybrid betagamma complex formed in the cell as determined by immunoprecipitation. These results suggest that different beta and gamma subunit types interact with each other with widely varying abilities, and this in combination with the level of expression of a subunit type in a mammalian cell determines which G protein will be active in that cell. The strong preference of all gamma subunit types for the beta1 subunit type explains the preponderence of this subunit type in most G proteins.

Animals↗

PubMed1995

A brain-specific G protein gamma subunit.

Two different cDNAs for G protein gamma subunits have been isolated from mouse brain. One encodes a novel gamma subunit, gamma 4, the expression of which is detected only in brain. A fragment of this cDNA had been isolated previously. The other cDNA encodes gamma 3, a subunit type previously isolated from bovine brain. The primary structure of the gamma 3 subunit is conserved completely across species indicating that the diversity in the structure of the gamma subunits is of functional consequence. Moreover, gamma 2, gamma 3 and gamma 4, which are predominantly expressed in brain, are more homologous to each other than other gamma subunits, indicating that the G protein gamma subunits belong to distinct subfamilies similar to the alpha subunits.

Amino Acid Sequence↗

PubMed1995

Efficient interaction with a receptor requires a specific type of prenyl group on the G protein gamma subunit.

Post-translational prenylation of the carboxyl-terminal cysteine is a characteristic feature of the guanine nucleotide-binding protein (G protein) gamma subunits. Recent findings show that the farnesylated COOH-terminal tail of the gamma 1 subunit is a specific determinant of rhodopsin-transducin coupling. We show here that when synthetic peptides specific to the COOH-terminal tail of gamma 1 are chemically modified with geranyl, farnesyl, or geranylgeranyl groups and tested for their ability to interact with light activated rhodopsin, the farnesylated peptide is significantly more effective. These results show that an appropriate isoprenoid on the G protein gamma subunit serves not only a membrane anchoring function but in combination with the COOH-terminal domain specifies receptor-G protein coupling.

Acyclic Monoterpenes↗