Reconstitution of receptors and GTP-binding regulatory proteins (G Proteins) in Sf9 Cells: a direct evaluation of selectivity in receptor.G protein coupling

Barr, A.J., Brass, L.F. and Manning, D.R. 1997. Reconstitution of receptors and GTP-binding regulatory proteins (G Proteins) in Sf9 Cells: a direct evaluation of selectivity in receptor.G protein coupling. Journal of Biological Chemistry. 272 (4), pp. 2223-2229.

TitleReconstitution of receptors and GTP-binding regulatory proteins (G Proteins) in Sf9 Cells: a direct evaluation of selectivity in receptor.G protein coupling
AuthorsBarr, A.J., Brass, L.F. and Manning, D.R.
Abstract

The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. α, β1, and γ2 subunits). Membranes were incubated with [35S]guanosine 5′-(3-O-thio)triphosphate (GTPγS) ± an agonist, and the amount of radiolabel bound to the α subunit was quantitated following immunoprecipitation. When expressed without receptor (but with β1γ2), the G protein subunits αz, α12, and α13 did not bind appreciable levels of [35S]GTPγS, consistent with a minimal level of GDP/[35S]GTPγS exchange. In contrast, the subunits αs and αq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPγS to αz but not to α12, α13, or αs. Binding to αz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPγS binding to αz and more robust increases in binding to αq, α12, and α13. Binding of [35S]GTPγS to αs was strongly enhanced only by the activated β1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.

JournalJournal of Biological Chemistry
Journal citation272 (4), pp. 2223-2229
ISSN0021-9258
Year24 Jan 1997
PublisherAmerican Society for Biochemistry and Molecular Biology
Digital Object Identifier (DOI)doi:10.1074/jbc.272.4.2223
Publication dates
Published24 Jan 1997

Related outputs

Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice
Hofmann, I., Geer, M.J., Vögtle, T., Crispin, A., Campagna, D.R., Barr, A.J., Calicchio, M.L., Heising, S., van Geffen, J.P., Kuijpers, M.J.E., Heemskerk, J.W.M., Eble, J.A., Schmitz-Abe, K., Obeng, E.A., Douglas, M., Freson, K., Pondarré, C., Favier, R., Jarvis, G.E., Markianos, K., Turro, E., Ouwehand, W.H., Mazharian, A., Fleming, M.D. and Senis, Y. 2018. Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice. Blood.

Targeting Receptor-Type Protein Tyrosine Phosphatases with Biotherapeutics: Is Outside-in Better than Inside-Out?
Senis, Y. A. and Barr, A.J. 2018. Targeting Receptor-Type Protein Tyrosine Phosphatases with Biotherapeutics: Is Outside-in Better than Inside-Out? Molecules. 23 (3), pp. 569-585.

Defining the molecular basis of interaction between R3 receptor-type protein tyrosine phosphatases and VE-cadherin
Dorofejeva, O. and Barr, A.J. 2017. Defining the molecular basis of interaction between R3 receptor-type protein tyrosine phosphatases and VE-cadherin. PLoS ONE. 12 (9 : e0184574.), pp. 1-23.

Automatic Selection of Molecular Descriptors using Random Forest: Application to Drug Discovery
Cano, G., Garcia-Rodriguez, J., Garcia-Garcia, A, Perez-Sanchez, H., Benediktsson, J.A., Thapa, A. and Barr, A.J. 2016. Automatic Selection of Molecular Descriptors using Random Forest: Application to Drug Discovery. Expert Systems with Applications. 72, pp. 151-159.

Targeting protein tyrosine phosphatase SHP2 for therapeutic intervention
Butterworth, S., Overduin, M. and Barr, A.J. 2014. Targeting protein tyrosine phosphatase SHP2 for therapeutic intervention. Future Medicinal Chemistry. 6 (12), pp. 1423-1437.

Recombinant baculovirus receptor systems to study receptor/G-protein communication
Windh, R., Barr, A.J. and Manning, D. 2000. Recombinant baculovirus receptor systems to study receptor/G-protein communication. in: Kenakin, T. and Angus, J. (ed.) The Pharmacology of Functional, Biochemical, and Recombinant Receptor Systems Berlin, Germany Springer-Verlag. pp. 335-362

Functional Studies On Receptor-Type Protein Tyrosine Phosphatases Of The R3 Subgroup Using Bimolecular Fluorescence Complementation (BiFC) Assays
Dorofejeva, O., Dwek, M. and Barr, A.J. 2014. Functional Studies On Receptor-Type Protein Tyrosine Phosphatases Of The R3 Subgroup Using Bimolecular Fluorescence Complementation (BiFC) Assays . Pharmacology 2014. London 16 Dec 2014 British Pharmacological Society.

Targeting protein tyrosine phosphatase SHP2 for therapeutic intervention
Butterworth, S., Overduin, M. and Barr, A.J. 2014. Targeting protein tyrosine phosphatase SHP2 for therapeutic intervention. Future Medicinal Chemistry. 6 (12), pp. 1423-1437.

Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states
Shintre, C.A., Pike, A.C.W., Li, Q., Kim, J.I., Barr, A.J., Goubin, S., Shrestha, L., Yang, J., Berridge, G., Ross, J., Stansfeld, P.J., Sansom, M.S.P., Edwards, A.M., Bountra, C., Marsden, B., von Delft, F., Bullock, A.N., Gileadi, O., Burgess-Brown, N.A. and Carpenter, E.P. 2013. Structures of ABCB10, a human ATP-binding cassette transporter in apo- and nucleotide-bound states. Proceedings of the National Academy of Sciences. 110 (24), pp. 9710-9715.

Receptor tyrosine phosphatase PTPγ is a regulator of spinal cord neurogenesis
Hashemia, H., Hurley, M., Gibson, A., Panova, V., Tchetchelnitski, V., Barr, A.J. and Stoker, A.W. 2011. Receptor tyrosine phosphatase PTPγ is a regulator of spinal cord neurogenesis. Molecular and Cellular Neuroscience. 46 (2), pp. 469-482.

Crystal structures of ABL-related gene (ABL2) in complex with imatinib, tozasertib (VX-680), and a type I inhibitor of the triazole carbothioamide class
Salah, E., Ugochukwu, E., Barr, A.J., von Delft, F., Knapp, S. and Elkins, J.M. 2011. Crystal structures of ABL-related gene (ABL2) in complex with imatinib, tozasertib (VX-680), and a type I inhibitor of the triazole carbothioamide class. Journal of Medicinal Chemistry. 54 (7), pp. 2359-2367.

Protein tyrosine phosphatases as drug targets: strategies and challenges of inhibitor development
Barr, A.J. 2010. Protein tyrosine phosphatases as drug targets: strategies and challenges of inhibitor development. Future Medicinal Chemistry. 2 (10), pp. 1563-1576.

CD148 enhances platelet responsiveness to collagen by maintaining a pool of active Src family kinases
Ellison, S., Mori, J., Barr, A.J. and Senis, Y.A. 2010. CD148 enhances platelet responsiveness to collagen by maintaining a pool of active Src family kinases. Journal of Thrombosis and Haemostasis. 8 (7), pp. 1575-1583.

Large-scale structural analysis of the classical human protein tyrosine phosphatome
Barr, A.J., Ugochukwu, E., Lee, W.H., King, O.N.F., Filippakopoulos, P., Alfano, I., Savitsky, P., Burgess-Brown, N.A., Muller, S. and Knapp, S. 2009. Large-scale structural analysis of the classical human protein tyrosine phosphatome. Cell. 136 (2), pp. 352-363.

HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain
Gingras, M.C., Zhang, Y.L., Kharitidi, D., Barr, A.J., Knapp, S., Tremblay, M.L. and Pause, A. 2009. HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain. PLoS ONE. Online.

Large scale structural analysis of protein tyrosine phosphatases
Barr, A.J. and Knapp, S. 2009. Large scale structural analysis of protein tyrosine phosphatases. in: Bradshaw, R. and Dennis, E. (ed.) Handbook of cell signaling (2nd edition) San Diego, CA Elsevier. pp. 871-876

Sequence-specific 1H, 13C and 15N backbone resonance assignments of the 34 kDa catalytic domain of human PTPN7
Jeeves, M., McClelland, D.M., Barr, A.J. and Overduin, M. 2008. Sequence-specific 1H, 13C and 15N backbone resonance assignments of the 34 kDa catalytic domain of human PTPN7. Biomolecular NMR Assignments. 2 (2), pp. 101-103.

MAPK-specific tyrosine phosphatases: new targets for drug discovery?
Barr, A.J. and Knapp, S. 2006. MAPK-specific tyrosine phosphatases: new targets for drug discovery? Trends in Pharmacological Sciences. 27 (10), pp. 525-530.

The crystal structure of human receptor protein tyrosine phosphatase κ phosphatase domain 1
Eswaran, J., Debreczeni, J.E., Longman, E., Barr, A.J. and Knapp, S. 2006. The crystal structure of human receptor protein tyrosine phosphatase κ phosphatase domain 1. Protein Science. 15 (6), pp. 1500-1505.

Crystal structure of human protein tyrosine phosphatase 14 (PTPN14) at 1.65-A resolution
Barr, A.J., Debreczeni, J.E., Eswaran, J. and Knapp, S. 2006. Crystal structure of human protein tyrosine phosphatase 14 (PTPN14) at 1.65-A resolution. Proteins. 63 (4), pp. 1132-1136.

Crystal structures and inhibitor identification for PTPN5, PTPRR and PTPN7: a family of human MAPK-specific protein tyrosine phosphatases
Eswaran, J., von Kries, J.P., Marsden, B., Longman, E., Debreczeni, J.E., Ugochukwu, E., Turnbull, A., Lee, W.H., Knapp, S. and Barr, A.J. 2006. Crystal structures and inhibitor identification for PTPN5, PTPRR and PTPN7: a family of human MAPK-specific protein tyrosine phosphatases. Biochemical Journal. 395 (3), pp. 483-491.

Phospholipase C-β 2 interacts with mitogen-activated protein kinase kinase 3
Barr, A.J., Marjoram, R.J., Xu, J. and Snyderman, R. 2002. Phospholipase C-β 2 interacts with mitogen-activated protein kinase kinase 3. Biochemical and Biophysical Research Communications. 293 (1), pp. 647-652.

RGS4 inhibits platelet-activating factor receptor phosphorylation and cellular responses
Richardson, R.M., Marjoram, R.J., Barr, A.J. and Snyderman, R. 2001. RGS4 inhibits platelet-activating factor receptor phosphorylation and cellular responses. Biochemistry. 40 (12), pp. 3583-3588.

Recombinant baculovirus receptor systems to study receptor/G-protein communication
Windh, R., Barr, A.J. and Manning, D.R. 2000. Recombinant baculovirus receptor systems to study receptor/G-protein communication. in: Kenakin, T. and Angus, J.A. (ed.) The Pharmacology of Functional, Biochemical, and Recombinant Receptor Systems Berlin Heidelberg Springer. pp. 335-362

Function and regulation of chemoattractant receptors
Haribabu, B., Richardson, R.M., Verghese, M.W., Barr, A.J., Zhelev, D.V. and Snyderman, R. 2000. Function and regulation of chemoattractant receptors. Immunologic Research. 22 (2-3), pp. 271-279.

Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits
Barr, A.J., Ali, H., Haribabu, B., Snyderman, R. and Smrcka, A.V. 2000. Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits. Biochemistry. 39 (7), pp. 1800-1806.

Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the Gi, Gq, and G12 families of heterotrimeric G proteins
Windh, R., Lee, M.J., Hla, T., An, S., Barr, A.J. and Manning, D.R. 1999. Differential coupling of the sphingosine 1-phosphate receptors Edg-1, Edg-3, and H218/Edg-5 to the Gi, Gq, and G12 families of heterotrimeric G proteins. Journal of Biological Chemistry. 274 (39), pp. 27351-27358.

Agonist-promoted GTP[S35]-binding as a probe for receptor.G protein communication in Sf9 cells
Barr, A.J. and Manning, D.R. 1999. Agonist-promoted GTP[S35]-binding as a probe for receptor.G protein communication in Sf9 cells. in: Manning, D.R. (ed.) G proteins: techniques of analysis Boca Raton, FL CRC Press. pp. 227-246

Differential regulation of formyl peptide and platelet-activating factor receptors: role of phospholipase Cbeta3 phosphorylation by protein kinase A
Ali, H., Sozzani, S., Fisher, I., Barr, A.J., Richardson, R.M., Haribabu, B. and Snyderman, R. 1998. Differential regulation of formyl peptide and platelet-activating factor receptors: role of phospholipase Cbeta3 phosphorylation by protein kinase A. Journal of Biological Chemistry. 273 (18), pp. 11012-11016.

Agonist-independent activation of Gz by the 5-hydroxytryptamine1A receptor co-expressed in spodoptera frugiperda cells: distinguishing inverse agonists from neutral antagonists
Barr, A.J. and Manning, D.R. 1997. Agonist-independent activation of Gz by the 5-hydroxytryptamine1A receptor co-expressed in spodoptera frugiperda cells: distinguishing inverse agonists from neutral antagonists. Journal of Biological Chemistry. 272 (52), pp. 32979-32987.

Protein kinase C mediates delayed inhibitory feedback regulation of human neurokinin type 1 receptor activation of phospholipase C in UC11 astrocytoma cells
Barr, A.J. and Watson, S.P. 1994. Protein kinase C mediates delayed inhibitory feedback regulation of human neurokinin type 1 receptor activation of phospholipase C in UC11 astrocytoma cells. Molecular Pharmacology. 46 (2), pp. 266-273.

Non-peptide antagonists, CP-96,345 and RP 67580, distinguish species variants in tachykinin NK1 receptors
Barr, A.J. and Watson, S.P. 1993. Non-peptide antagonists, CP-96,345 and RP 67580, distinguish species variants in tachykinin NK1 receptors. British Journal of Pharmacology. 108 (1), pp. 223-227.

The presence of NK3 tachykinin receptors on rat uterus
Barr, A.J., Watson, S.P., Bernal, A.L. and Nimmo, A.J. 1991. The presence of NK3 tachykinin receptors on rat uterus. European Journal of Pharmacology. 203 (2), pp. 287-290.

Permalink - https://westminsterresearch.westminster.ac.uk/item/94798/reconstitution-of-receptors-and-gtp-binding-regulatory-proteins-g-proteins-in-sf9-cells-a-direct-evaluation-of-selectivity-in-receptor-g-protein-coupling


Share this
Tweet
Email