Site-specific antibodies directed against G protein .beta. and .gamma. subunits: effects on .alpha. and .beta..gamma. subunit interaction

Murakami, Takeshi; Simonds, William F.; Spiegel, Allen M.

doi:10.1021/bi00126a009

Cited by 35 publications

(18 citation statements)

References 47 publications

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“…Primary antibodies used were: affinity-purified rabbit ATDG polyclonal antibody against N terminus of G␤ 5 (11), rabbit polyclonal antibody SGS against the C terminus of G␤ 5 (5), goat anti-RGS7 C-19 and R-20 IgG (Santa Cruz), rabbit anti-TFIID (TBP) N-12 IgG (Santa Cruz), anti-GFP monoclonal antibody (CLONTECH), and affinity-purified rabbit KT polyclonal antibody against the N terminus of G␤ 1 (21). Immunoprecipitation from brain nuclear extracts or PC12 cell lysates employed goat anti-RGS7 C-19 or normal goat IgG following the previously described methodology (11).…”

Section: Methodsmentioning

confidence: 99%

Nuclear Localization of G Protein β5 and Regulator of G Protein Signaling 7 in Neurons and Brain

Zhang

Barr

et al. 2001

Journal of Biological Chemistry

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The role that G␤ 5 regulator of G protein signaling (RGS) complexes play in signal transduction in brain remains unknown. The subcellular localization of G␤ 5 and RGS7 was examined in rat PC12 pheochromocytoma cells and mouse brain. Both nuclear and cytosolic localization of G␤ 5 and RGS7 was evident in PC12 cells by immunocytochemical staining. Subcellular fractionation of PC12 cells demonstrated G␤ 5 immunoreactivity in the membrane, cytosolic, and nuclear fractions. Analysis by limited proteolysis confirmed the identity of G␤ 5 in the nuclear fraction. Subcellular fractionation of mouse brain demonstrated G␤ 5 and RGS7 but not G␥ 2/3 immunoreactivity in the nuclear fraction. RGS7 and G␤ 5 were tightly complexed in the brain nuclear extract as evidenced by their coimmunoprecipitation with anti-RGS7 antibodies. Chimeric protein constructs containing green fluorescent protein fused to wild-type G␤ 5 but not green fluorescent fusion proteins with G␤ 1 or a mutant G␤ 5 impaired in its ability to bind to RGS7 demonstrated nuclear localization in transfected PC12 cells. These findings suggest that G␤ 5 undergoes nuclear translocation in neurons via an RGS-dependent mechanism. The novel intracellular distribution of G␤ 5 ⅐RGS protein complexes suggests a potential role in neurons communicating between classical heterotrimeric G protein subunits and/or their effectors at the plasma membrane and the cell nucleus.Seven transmembrane-spanning receptors respond to extracellular signals and in turn regulate intracellular processes through their interaction with signal-transducing heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) in eukaryotic cells (1). Complementary DNAs from five G protein ␤ subunit genes (G␤ 1-5 ) have been identified by molecular cloning. Whereas the G␤ 1-4 isoforms are highly homologous (80 -90%) and widely expressed (2), the G␤ 5 isoform exhibits much less homology with other isoforms (ϳ50%) and is preferentially expressed in brain (3). A splice variant of G␤ 5 , G␤ 5 -long (G␤ 5 L), is present in retina, which contains a 42-amino acid N-terminal extension (4).Although G␤ 5 can be shown to interact with classical components of heterotrimeric G proteins signaling pathways such as G␥2 (3, 5, 6), G␣q (6), and phospholipase C-␤ (3, 5, 7, 8) when tested in vitro, no such interactions have been demonstrated in native tissues. Instead, G␤ 5 has been purified from retinal cytosol bound to regulator of G protein signaling (RGS) 1 protein-7 (RGS7) (9, 10) and from brain bound to RGS6 (11) and RGS7 (10, 11). Additionally, a tight native complex between G␤ 5 L and RGS9 was isolated from retinal rod outer segment membrane extracts (12). Studies of recombinant proteins in vitro show that a tight interaction with RGS proteins 6, 7, and 11 is demonstrable for G␤ 5 and G␤ 5 L but not for the other G␤ isoforms, mediated by a G␥-like (GGL) domain present in a subfamily of RGS proteins (13-16). These recent novel observations underscore the view of G␤ 5 as a unique and highly specialized G prote...

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Section: Methodsmentioning

confidence: 99%

Nuclear Localization of G Protein β5 and Regulator of G Protein Signaling 7 in Neurons and Brain

Zhang

Barr

et al. 2001

Journal of Biological Chemistry

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“…Furthermore, mutation of Cys-215 of Go a prevents cross-linking to 0-y, although the mutant a subunit still binds to 13-y (38). In addition, several regions of transducin 1 have been implicated in the a-,8 interaction, because antibodies raised to peptides defining the amino t NRCC publication 36165. terminus, codons 127 to 136 and codons 256 to 265, of transducin 131 block interaction with transducin a (25). These results provide a general map of potential sites of a-, interactions but lack the resolution that can be provided by a mutant selection approach.…”

mentioning

confidence: 87%

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

Whiteway¹,

Clark²,

Leberer³

et al. 1994

Mol. Cell. Biol.

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The GPAI, STE4, and STE18 genes of Saccharomyces cerevisiae encode the a, jI, and y subunits, respectively, of a G protein involved in the mating response pathway. We have found that mutations G124D, W136G, W136R, and AL138 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 Gpal protein. A mutation at position E307 of the Gpal protein both suppresses the constitutive signaling phenotype of some mutant Ste4 proteins and allows the mutant a subunit to physically associate with a specific mutant GoI subunit. The mutation in the Gpal 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 a subunit with the wild-type ,1 subunit. Yeast cells constructed to contain only the mutant a and , subunits mate and respond to pheromones, although they exhibit partial induction of the pheromone response pathway. Because the ability of the modified Gat 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.Heterotrimeric G proteins function to transmit signals from a variety of cell surface receptor proteins to intracellular signaling components through a common molecular mechanism (32). The at and 13y subunits of heterotrimeric G proteins undergo an association-dissociation cycle in response to the interaction between the cell surface receptor and the receptor's cognate ligand. This association-dissociation cycle is regulated by guanine nucleotides. Unstimulated receptors are coupled to nonactivated G proteins in which the subunits are associated, and GDP is bound to the a subunit. The ligandbound receptor stimulates exchange of the bound GDP for GTP, and this exchange triggers subunit dissociation; subsequent GTP hydrolysis allows reassociation of the subunits and a return to the resting state. This cycle predicts that the at subunit of G proteins is capable of undergoing a conformational change which modifies its affinity for the 13y subunit (9).We have used a genetic approach to identify specific regions of the G protein a and 1 subunits that are important for their proper association with each other. The strategy of defining molecular interactions through genetics can supplement or even correct direct structural studies and can provide a high level of resolution (13,33,45). Previous approaches to investigating the associations among G-protein subunits have shown that deletion of the amino terminus of Gao blocks association of the Ga and GP subunits (10), while mutations in the switch box of Gs ax prevent a-1 dissociation in response to ligand stimulation of receptor (23). Furthermore, mutation of Cys-215 of Go a prevents cross-linking to 0-y, although ...

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“…First, proteins such as ~-transducin and Secl3, which are known to interact biochemically with other proteins, consist mainly of WD repeats, suggesting that binding might occur through the repeats (for review, see Conklin and Bourne 1993;Salama et al 1993). Second, antibodies raised to peptides within the repeats of [3-transducin can inhibit its ability to interact with the transducin ~ subunit (Murakami et al 1992). Third, Gpal and Ste4, the c~ and B subunits of a yeast G protein, interact in vivo in the two-hybrid fusion assay; this interaction is disrupted by mutations in the second WD repeat of Ste4 (Clark et al 1993;Whiteway et al 1994).…”

Section: A Single Wd Repeat Is a Protein-protein Interaction Domainmentioning

confidence: 99%

The WD repeats of Tup1 interact with the homeo domain protein alpha 2.

Komachi¹,

Redd²,

Johnson³

1994

Genes Dev.

207

214

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Tupl and Ssn6 transcriptionally repress a wide variety of genes in yeast but do not appear to bind DNA. We provide genetic and biochemical evidence that the DNA-binding protein a2, a regulator of cell-type-specific genes, recruits the Tupl/Ssn6 repressor by directly interacting with Tupl. This interaction is mediated by a region of Tupl containing seven copies of the WD repeat, a 40 amino acid motif of unknown function found in many other proteins. We have found that a single WD repeat will interact with a2, indicating that the WD repeat is a protein-protein interaction domain. Furthermore, a fragment of Tupl containing primarily WD repeats provides at least partial repression in the absence of Ssn6, suggesting that the repeats also mediate interaction between Tupl and other components of the repression machinery.

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Site-specific antibodies directed against G protein .beta. and .gamma. subunits: effects on .alpha. and .beta..gamma. subunit interaction

Cited by 35 publications

References 47 publications

Nuclear Localization of G Protein β5 and Regulator of G Protein Signaling 7 in Neurons and Brain

Nuclear Localization of G Protein β5 and Regulator of G Protein Signaling 7 in Neurons and Brain

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

The WD repeats of Tup1 interact with the homeo domain protein alpha 2.

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