Role of the Prenyl Group on the G Protein γ Subunit in Coupling Trimeric G Proteins to A1 Adenosine Receptors*

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein α subunits and by the composition of the βγ dimers. To determine the role of the γ subunit prenyl modification in this interaction, the CaaX motifs in the γ1 and γ2 subunits were altered to direct modification with different prenyl groups, recombinant βγ dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the βγ dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the α subunit. The β1γ1 dimer (modified with farnesyl) was significantly less effective than β1γ2 (modified with geranylgeranyl) in either assay. The β1γ1-S74L dimer (modified with geranylgeranyl) was nearly as effective as β1γ2 in either assay. The β1γ2-L71S dimer (modified with farnesyl) was significantly less active than β1γ2. Using 125I-labeled βγ subunits, it was determined that native and altered βγ dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the γ subunit is an important determinant of the interaction between receptors and G protein γ subunits.

[1]  M. Lindorfer,et al.  [15] Expression and purification of G protein βγ subunits using baculovirus expression system , 1996 .

[2]  D. Hunt,et al.  G Protein γ Subunits with Altered Prenylation Sequences Are Properly Modified When Expressed in Sf9 Cells* , 1996, The Journal of Biological Chemistry.

[3]  H. Hamm,et al.  The 2.0 Å crystal structure of a heterotrimeric G protein , 1996, Nature.

[4]  Y. Fukada,et al.  Primary Structure of a γ Subunit of G Protein, γ12, and Its Phosphorylation by Protein Kinase C * , 1995, The Journal of Biological Chemistry.

[5]  S. Sprang,et al.  The structure of the G protein heterotrimer Giα1 β 1 γ 2 , 1995, Cell.

[6]  N. Gautam,et al.  Efficient Interaction with a Receptor Requires a Specific Type of Prenyl Group on the G Protein γ Subunit (*) , 1995, The Journal of Biological Chemistry.

[7]  M. Ermolaeva,et al.  Receptor-G protein coupling is established by a potential conformational switch in the beta gamma complex. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[8]  C. Kunsch,et al.  Isolation of cDNA Clones Encoding Eight Different Human G Protein γ Subunits, Including Three Novel Forms Designated the γ4, γ10, and γ11 Subunits (*) , 1995, The Journal of Biological Chemistry.

[9]  P. Molinoff,et al.  Expression of the Human 5-Hydroxytryptamine1A Receptor in Sf9 Cells , 1995, The Journal of Biological Chemistry.

[10]  S. Pfeffer,et al.  Rab GDP Dissociation Inhibitor: Putting Rab GTPases in the Right Place (*) , 1995, The Journal of Biological Chemistry.

[11]  R. Kahn,et al.  The Myristoylated Amino Terminus of ADP-ribosylation Factor 1 Is a Phospholipid- and GTP-sensitive Switch (*) , 1995, The Journal of Biological Chemistry.

[12]  D. Bok,et al.  Molecular Cloning and Characterization of the G Protein γ Subunit of Cone Photoreceptors (*) , 1995, The Journal of Biological Chemistry.

[13]  N. Ryba,et al.  A Novel GTP-binding Protein γ-Subunit, Gγ8, Is Expressed during Neurogenesis in the Olfactory and Vomeronasal Neuroepithelia (*) , 1995, The Journal of Biological Chemistry.

[14]  E. Neer.  Heterotrimeric C proteins: Organizers of transmembrane signals , 1995, Cell.

[15]  Henry R. Bourne,et al.  Lipid Modifications of Trimeric G Proteins (*) , 1995, The Journal of Biological Chemistry.

[16]  Y. Fukada,et al.  Characterization of interactions between transducin alpha/beta gamma-subunits and lipid membranes. , 1994, The Journal of biological chemistry.

[17]  N. Gautam,et al.  A farnesylated domain in the G protein gamma subunit is a specific determinant of receptor coupling. , 1994, The Journal of biological chemistry.

[18]  P. Sternweis.  The active role of ? in signal transduction , 1994 .

[19]  D. Clapham,et al.  Recombinant G-protein βγ-subunits activate the muscarinic-gated atrial potassium channel , 1994, Nature.

[20]  A. Gilman,et al.  G protein βγ subunits: Simplified purification and properties of novel isoforms , 1994 .

[21]  T. K. Harden,et al.  Selective activation of phospholipase C by recombinant G-protein alpha- and beta gamma-subunits. , 1994, The Journal of biological chemistry.

[22]  N. Gautam,et al.  Specific interaction with rhodopsin is dependent on the gamma subunit type in a G protein. , 1993, The Journal of biological chemistry.

[23]  M. Lohse,et al.  Specific enhancement of beta-adrenergic receptor kinase activity by defined G-protein beta and gamma subunits. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[24]  David E. Clapham,et al.  New roles for G-protein (βγ-dimers in transmembrane signalling , 1993, Nature.

[25]  J. Wess.  Molecular basis of muscarinic receptor function , 1993 .

[26]  A. Gilman,et al.  The importance of G-protein βλ subunits , 1993 .

[27]  Bruce R. Conklin,et al.  Structural elements of Gα subunits that interact with Gβγ, receptors, and effectors , 1993, Cell.

[28]  C J Marshall,et al.  Protein prenylation: a mediator of protein-protein interactions. , 1993, Science.

[29]  G. Schultz,et al.  Selectivity in signal transduction determined by gamma subunits of heterotrimeric G proteins. , 1993, Science.

[30]  L. Birnbaumer,et al.  Receptor-to-effector signaling through G proteins: Roles for βγ dimers as well as α subunits , 1992, Cell.

[31]  P. Casey,et al.  Biochemistry of protein prenylation. , 1992, Journal of lipid research.

[32]  M. Simon,et al.  G protein beta gamma subunits synthesized in Sf9 cells. Functional characterization and the significance of prenylation of gamma. , 1992, The Journal of biological chemistry.

[33]  L. Weinstein,et al.  Receptor-effector coupling by G proteins: implications for normal and abnormal signal transduction. , 1992, Endocrine reviews.

[34]  G. Schultz,et al.  Different β-subunits determine G-protein interaction with transmembrane receptors , 1992, Nature.

[35]  V. Kalman-Maltese,et al.  Expression of functional G protein beta gamma dimers of defined subunit composition using a baculovirus expression system. , 1992, The Journal of biological chemistry.

[36]  A. Tucker,et al.  Cloning and expression of a bovine adenosine A1 receptor cDNA , 1992, FEBS letters.

[37]  R. Figler,et al.  Expression and purification of functional G protein alpha subunits using a baculovirus expression system. , 1992, The Journal of biological chemistry.

[38]  Y. Fukada,et al.  Carboxyl methylation and farnesylation of transducin gamma‐subunit synergistically enhance its coupling with metarhodopsin II. , 1991, The EMBO journal.

[39]  G. Schultz,et al.  Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium currents , 1991, Nature.

[40]  H. Tamir,et al.  Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms. , 1991, The Journal of biological chemistry.

[41]  A. Chiu,et al.  DuP 753 can antagonize the effects of angiotensin II in rat liver. , 1991, Molecular pharmacology.

[42]  W. Simonds,et al.  G-protein beta gamma dimers. Membrane targeting requires subunit coexpression and intact gamma C-A-A-X domain. , 1991, The Journal of biological chemistry.

[43]  C. Taylor,et al.  The role of G proteins in transmembrane signalling. , 1990, The Biochemical journal.

[44]  F. Cañada,et al.  The gamma subunit of transducin is farnesylated. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Y. Fukada,et al.  Farnesylated γ-subunit of photoreceptor G protein indispensable for GTP-binding , 1990, Nature.

[46]  M. Gelb,et al.  Brain G protein gamma subunits contain an all-trans-geranylgeranylcysteine methyl ester at their carboxyl termini. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[47]  P. Casey,et al.  G protein gamma subunits contain a 20-carbon isoprenoid. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[48]  P. Sternweis,et al.  Reconstitution of resolved muscarinic cholinergic receptors with purified GTP-binding proteins. , 1985, The Journal of biological chemistry.

[49]  J. Linden,et al.  [125I]Aminobenzyladenosine, a new radioligand with improved specific binding to adenosine receptors in heart. , 1985, Circulation research.

[50]  B. K. Fung.  Characterization of transducin from bovine retinal rod outer segments. I. Separation and reconstitution of the subunits. , 1983, The Journal of biological chemistry.

[51]  J. Morrissey,et al.  Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. , 1981, Analytical biochemistry.

[52]  P. Fraker,et al.  Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. , 1978, Biochemical and biophysical research communications.

[53]  J. Exton.  Phosphoinositide phospholipases and G proteins in hormone action. , 1994, Annual review of physiology.

[54]  C. Strader,et al.  Structure and function of G protein-coupled receptors. , 1994, Annual review of biochemistry.

[55]  M. Camps,et al.  Stimulation of phospholipase C‐β2 by recombinant guanine‐nucleotide‐binding protein βγ dimers produced in a baculovirus/insect cell expression system , 1994 .

[56]  D. Knighton,et al.  Crystal structures of the catalytic subunit of cAMP-dependent protein kinase reveal general features of the protein kinase family. , 1993, Receptor.

[57]  A. Gilman,et al.  Influence of gamma subunit prenylation on association of guanine nucleotide-binding regulatory proteins with membranes. , 1992, Molecular biology of the cell.

[58]  J. Thorner,et al.  Model systems for the study of seven-transmembrane-segment receptors. , 1991, Annual review of biochemistry.

[59]  L. Birnbaumer,et al.  G proteins in signal transduction. , 1990, Annual review of pharmacology and toxicology.

[60]  A. Gilman,et al.  G proteins: transducers of receptor-generated signals. , 1987, Annual review of biochemistry.