[39] Preparation, characterization, and use of antibodies with specificity for G-protein y subunits

Robishaw, Janet D.; Balcueva, Eric A.

doi:10.1016/s0076-6879(94)37086-9

Cited by 15 publications

(15 citation statements)

References 12 publications

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“…Recombinant viruses were identified by immunoblotting Sf9 cell lysates for expression of the appropriate subunits. Subtype-specific antibodies were generated as described previously (20) using the following synthetic peptides: ␥ 4 , CKEGMSNNSTTSIS (amino acids 2-14); ␥ 10 , CKDALLVGVPAGSNPFREPR (amino acids 45-63); and ␥ 11 , CPALHIEDLPEK (amino acids 2-12). Other subtype-specific antibodies have been described previously (20,21).…”

Section: Methodsmentioning

confidence: 99%

“…Subtype-specific antibodies were generated as described previously (20) using the following synthetic peptides: ␥ 4 , CKEGMSNNSTTSIS (amino acids 2-14); ␥ 10 , CKDALLVGVPAGSNPFREPR (amino acids 45-63); and ␥ 11 , CPALHIEDLPEK (amino acids 2-12). Other subtype-specific antibodies have been described previously (20,21). Recombinant virus encoding G␣ i1 containing a hexahistidine tag at amino acid position 121 was kindly provided by Dr. T. Kozasa (Southwestern Medical Center, Dallas, TX).…”

Section: Methodsmentioning

confidence: 99%

“…The identity of each ␤␥ purified preparation was confirmed by immunoblotting with antibodies specific for each ␥ subtype. Antibodies specific for the ␥ 1 , ␥ 2 , ␥ 3 , ␥ 5 , and ␥ 7 subunits were used for this purpose previously (20). However, antibodies specific for the newly described ␥ 4 , ␥ 10 , and ␥ 11 subunits needed to be generated against synthetic peptides based on the unique amino acid sequences of these proteins.…”

Section: Methodsmentioning

confidence: 99%

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The α2A-Adrenergic Receptor Discriminates between Gi Heterotrimers of Different βγ Subunit Composition in Sf9 Insect Cell Membranes

Richardson

Robishaw

1999

Journal of Biological Chemistry

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In view of the expanding roles of the ␤␥ subunits of the G proteins in signaling, the possibility was raised that the rich diversity of ␤␥ subunit combinations might contribute to the specificity of signaling at the level of the receptor. To test this possibility, Sf9 cell membranes expressing the recombinant ␣ 2A -adrenergic receptor were used to assess the contribution of the ␤␥ subunit composition. Reconstituted coupling between the receptor and heterotrimeric G i protein was assayed by high affinity, guanine nucleotide-sensitive binding of the ␣ 2 -adrenergic agonist, [3 H]UK-14,304. Supporting this hypothesis, the present study showed clear differences in the abilities of the various ␤␥ dimers, including those containing the ␤ 3 subtype and the newly described ␥ 4 , ␥ 10 , and ␥ 11 subtypes, to promote interaction of the same ␣ i subunit with the ␣ 2A -adrenergic receptor.Consistent with the steadily increasing number of G protein 1 ␤ and ␥ subtypes that has been revealed in recent years (1), in vivo studies have indicated a role for this structural diversity in the specificity of signaling. In this regard, antisense studies by Kleuss et al. (2,3) have demonstrated a specific requirement for the ␤ 1 and ␥ 3 subunits in the somatostatin receptor signaling pathway in rat pituitary GH 3 cells, with a similarly specific requirement for the ␤ 3 and ␥ 4 subunits in the muscarinic receptor signaling pathway. Also, a ribozyme study by Wang et al. (4) has shown a specific involvement of the ␥ 7 subunit in the ␤-adrenergic receptor signaling pathway in human kidney 293 cells. Taken together, these in vivo studies indicate that the composition of the ␤␥ dimer has important ramifications for the fidelity of signaling that is probably manifested at the level of the receptor.A growing body of in vitro evidence supports a direct interaction between the receptor and the ␤␥ dimer (5). In particular, direct interaction of transducin ␤␥ with rhodopsin has been shown with a fluorescence energy transfer technique (6). This association was blocked by a synthetic peptide derived from the carboxyl-terminal tail of rhodopsin, suggesting a site of direct contact between ␤␥ and rhodopsin. Moreover, cross-linking studies have confirmed a receptor contact site on the ␤ subunit. A synthetic peptide derived from the carboxyl-terminal portion of the putative third cytoplasmic loop of the ␣ 2A -AR could be cross-linked to the carboxyl-terminal region of the ␤ subunit (7).To date, in vitro studies examining the contribution of a limited number of ␤␥ dimers to the specificity of receptor coupling have not yielded the same high degree of discrimination shown in the in vivo studies cited above (8, 9). The present study extended this analysis to the ␣ 2A -adrenergic receptor and to ␤␥ dimers that represent the most extensive degree of structural diversity examined to date. Since baculovirus expression has been shown to be an effective means for producing functional G protein subunits (10 -13) as well as G protein-coupled receptors (8,9,14,15)...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The α2A-Adrenergic Receptor Discriminates between Gi Heterotrimers of Different βγ Subunit Composition in Sf9 Insect Cell Membranes

Richardson

Robishaw

1999

Journal of Biological Chemistry

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“…The concentrations of the cholate-solubilized proteins were determined by Amido Black assay (37). Equal amounts of cholate-solubilized proteins were resolved on 15% SDS-polyacrylamide gels and transferred to Nitro-plus nitrocellulose (0.45-m pore size; Micron Separations Inc.), using a high temperature procedure (38). Following transfer, the nitrocellulose blots were probed with G protein-specific, primary antibodies (23,25,38) and 125 I-labeled goat anti-rabbit F(abЈ) 2 fragment, secondary antibody (1 ϫ 10 5 dpm/ml; PerkinElmer Life Sciences) in high detergent blotto (38).…”

Section: Generation Of Hek 293 Cells Stably Expressing Either the D 1mentioning

confidence: 99%

“…Equal amounts of cholate-solubilized proteins were resolved on 15% SDS-polyacrylamide gels and transferred to Nitro-plus nitrocellulose (0.45-m pore size; Micron Separations Inc.), using a high temperature procedure (38). Following transfer, the nitrocellulose blots were probed with G protein-specific, primary antibodies (23,25,38) and 125 I-labeled goat anti-rabbit F(abЈ) 2 fragment, secondary antibody (1 ϫ 10 5 dpm/ml; PerkinElmer Life Sciences) in high detergent blotto (38). After washing, the blots were subjected to autoradiography by exposure to BioMax MS film (Eastman Kodak Co.), and the intensities of the immunodetectable bands were quantified using a PhosphorImager SI (Molecular Dynamics Co.).…”

Section: Generation Of Hek 293 Cells Stably Expressing Either the D 1mentioning

confidence: 99%

Differential Dependence of the D1 and D5Dopamine Receptors on the G Protein γ7 Subunit for Activation of Adenylylcyclase

Wang¹,

Jolly²,

Surmeier³

et al. 2001

Journal of Biological Chemistry

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The D 1 dopamine receptor, G protein ␥ 7 subunit, and adenylylcyclase are selectively expressed in the striatum, suggesting their potential interaction in a common signaling pathway. To evaluate this possibility, a ribozyme strategy was used to suppress the expression of the G protein . Studies suggest that imbalances between these two opposing classes lead to deficiencies in movement and cognitive performance (2, 3). In particular, alterations in the D 1 -like dopamine receptors are implicated in a variety of neurologic and psychiatric disorders, such as Parkinson's disease, Tourette's syndrome, and schizophrenia. Thus, achieving a better understanding of the D 1 -like dopamine receptors and the signaling pathways they activate may suggest more selective therapeutic targets in these diseases.The D 1 and D 5 dopamine receptors stimulate adenylylcyclase activity through their coupling to heterotrimeric G proteins (1, 4 -6). Because the function of these heterotrimeric G proteins was originally ascribed to the ␣ subunit, most research has focused on determining its identity. Of the several ␣ subunits identified to date, reconstitution studies have shown that coupling of D 1 dopamine receptors to adenylylcyclase can be mediated only by the ␣ s and ␣ olf subunits of the G s subclass (4, 5, 7-9). Although sharing 88% amino acid homology, the ␣s and ␣ olf subunits show very divergent expression patterns, ranging from the ubiquitous expression of the ␣ s subunit to the olfactory and neuron-specific expression of the ␣ olf subunit (10). Immunoprecipitation studies (11) have confirmed the interaction between the ␣ s subunit and the D 1 dopamine receptors in cells, whereas in situ hybridization (12, 13) and gene targeting (14) studies have suggested a possible interaction between the ␣ olf subunit and the D 1 dopamine receptor in striatum.By contrast, little effort has focused on determining the identity of the ␤␥ subunits of the G protein despite mounting evidence for their importance in receptor recognition (15,16). Of particular interest, reconstitution studies (17-21) and reverse genetic approaches (22,23) have shown that the nature of the ␥ subunit is an important determinant of its interaction with receptor. Consistent with such a role, 12 ␥ subunit genes have been identified that show extensive structural diversity (24). Recently, we used a ribozyme approach to begin to elucidate their functions (23,25,26). This approach identified the ␥ 7 subunit as a specific component of the G protein that couples the ␤-adrenergic receptor, but not the prostaglandin E 1 receptor, to stimulation of adenylylcyclase in HEK 293 cells (23). Although expressed in a variety of tissues and cell types (27,28), the ␥ 7 subunit expression is most abundant in medium spiny neurons in striatum (13). This pattern of expression is shared by the D 1 dopamine receptor and adenylylcyclase (4, 12, 29 -31), raising the possibility that all of these components may be involved in the same signaling pathway. In the present study, we used the ribozyme ...

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Human G Protein γ11 and γ14 Subtypes Define a New Functional Subclass

Balcueva

Wang

Hughes

et al. 2000

Experimental Cell Research

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[39] Preparation, characterization, and use of antibodies with specificity for G-protein y subunits

Cited by 15 publications

References 12 publications

The α2A-Adrenergic Receptor Discriminates between Gi Heterotrimers of Different βγ Subunit Composition in Sf9 Insect Cell Membranes

The α2A-Adrenergic Receptor Discriminates between Gi Heterotrimers of Different βγ Subunit Composition in Sf9 Insect Cell Membranes

Differential Dependence of the D1 and D5Dopamine Receptors on the G Protein γ7 Subunit for Activation of Adenylylcyclase

Human G Protein γ11 and γ14 Subtypes Define a New Functional Subclass

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