Alan Wise

Agonist Occupation of an α2A-Adrenoreceptor-Gi1α Fusion Protein Results in Activation of Both Receptor-linked and Endogenous Gi Proteins COMPARISONS OF THEIR CONTRIBUTIONS TO GTPase ACTIVITY AND SIGNAL TRANSDUCTION AND ANALYSIS OF RECEPTOR-G PROTEIN ACTIVATION STOICHIOMETRY

A fusion protein between a pertussis toxin-resistant (C351G) mutant of the α subunit of the G protein Gi1 and the porcine α2A-adrenoreceptor was stably expressed in Rat 1 fibroblasts. Agonists caused stimulation of high affinity GTPase activity, which was partially prevented by pertussis toxin treatment, demonstrating that the toxin-resistant component of the GTPase activity was derived from the receptor-fused G protein and the remainder from endogenous Giα. Half-maximal stimulation of the GTPase activity of endogenous Gi was achieved with lower concentrations of agonist. Although the K m for GTP of the fusion protein-linked Gi was lower than for the endogenous G protein, V max measurements demonstrated that adrenaline activated some 5 mol of endogenous Gi/mol of fusion protein-linked Gi. The isolated α2A-adrenoreceptor could activate Gs; however, the fusion protein did not. Compared with adrenaline, the efficacy of a range of partial agonists to stimulate endogenous Giα was greater than for the fusion protein-constrained C351G Gi1α. α2A-Adrenoreceptor agonists could stimulate both p44 mitogen-activated protein kinase and p70 S6 kinase and inhibit forskolin-amplified adenylyl cyclase activity in untreated α2A-adrenoreceptor-C351G Gi1α fusion protein-expressing cells, but these signals were abolished following pertussis toxin treatment. These results demonstrate conclusively, and for the first time, that agonist occupancy of a receptor-G protein fusion protein can result in activation of G proteins other than that physically linked to the receptor. This was selective between G protein classes. Analysis of the contributions of fusion protein-linked and endogenous G proteins to agonist-stimulated GTPase activity provided a direct and original measure of receptor-G protein activation stoichiometry.

Rescue of Functional Interactions between the α2A-Adrenoreceptor and Acylation-resistant Forms of Gi1α by Expressing the Proteins from Chimeric Open Reading Frames

Co-expression of the α2A-adrenoreceptor with a pertussis toxin-resistant (C351G), but not with an also palmitoylation-resistant (C3S/C351G), form of the α subunit of Gi1 resulted in agonist-induced, pertussis toxin-independent, GTP hydrolysis. Construction and expression of a chimeric fusion protein between the receptor and C351G Gi1α generated a membrane protein in which the G protein element was activated by receptor agonist. An equivalent fusion protein containing C3S/C351G Gi1α rescued the ability of receptor agonist to activate this mutant. Fusion proteins of a palmitoylation-resistant (C442A) α2A-adrenoreceptor and either C351G or C3S/C351G Gi1α also responded effectively to agonist. Myristoylation resistant (G2A/C351G) and combined acylation-resistant (G2A/C3S/C351G) mutants of Gi1α are cytosolic proteins. Expression of these as chimeric α2A-adrenoreceptor-G protein fusions restored membrane localization and activation of the G protein by receptor agonist. These studies demonstrate the general utility of generating chimeric fusion proteins to examine receptor regulation of G protein function and that the lack of functional activation of acylation-negative G proteins by a co-expressed receptor is related to deficiencies in cellular targeting and location rather than an inherent incapacity to produce appropriate protein-protein interactions and signal transmission.

Measurement of agonist efficacy using an α2A-adrenoceptor-Gi1α fusion protein

A fusion protein was constructed between the porcine α2A‐adrenoceptor and a pertussis toxin‐insensitive (Cys351Gly) form of the α subunit of the G protein Gi1. Addition of agonist ligands to membranes of COS‐7 cells transiently transfected to express this construct, and treated with pertussis toxin prior to cell harvest, resulted in stimulation of both high affinity GTPase activity and enhanced binding of [35S]GTPγS. By considering the fusion protein as an agonist‐activated enzyme and measuring V max of GTP hydrolysis a range of agonist ligands displayed varying efficacy in their capacity to activate the receptor‐associated G protein with adrenaline=noradrenaline=α‐methylnoradrenaline>UK14304>BHT933≥xylazine=clonidine. A similar rank order was observed following independent co‐expression of the α2A‐adrenoceptor and Cys351Gly‐Gi1α. These data demonstrate the utility and applicability of using a receptor‐G protein fusion protein approach, in which the stoichiometry of receptor and G protein is fixed at 1:1, to measure and further understand the nature of agonist efficacy.

Interaction of the G-protein G11α with receptors and phosphoinositidase C:

Wild‐type and palmitoylation defective mutants of the murine G protein G11α were transfected into HEK293 cells. Wild‐type G11α was membrane associated, Cys9Ser Cys10Ser G11α was present in the soluble fraction whilst both Cys9Ser G11α and Cys10Ser G11α were distributed between the fractions. Expression of the rat TRH receptor resulted in agonist stimulation of inositol phosphate accumulation. The degree of stimulation produced by TRH following co‐transfection of the palmitoylation‐resistant forms of G11α compared to the wild‐type protein correlated with the amount of membrane‐associated G protein.

Expression of the Arabidopsis G-protein GPα1: purification and characterisation of the recombinant protein

The Arabidopsis Gα subunit, GPα1, was expressedwithin Escherichia coli by co-transformation with the expressionvector and the dnaY gene which encodes tRNAArgAGA/AGG. Isolation of the recombinant GPα1 in a highly pureform could be achieved by a combination of anion exchange and dyeaffinity chromatography or by a single step affinity procedure viachromatography on 4-amino-anilido-GTP agarose. The recombinant proteinyielded by both procedures was highly active and bound GTPγS withan apparent Kd in the nM range. GTPγS binding wasstimulated two-fold in the presence of Zn2+ compared with that inthe presence of Mg2+, Mn2+ or Ca2+.Abbreviations: 4aaGTP, 4-amino-anilido-GTP; GTPγS,guanosine- 5′-(3-O-thiotriphosphate), PMSF,phenylmethylsulphonyl fluoride; PVDF, polvinylidene fluoride;rGPα1, recombinant GPα1

Expression of the Human β2‐Adrenoceptor in NCB20 Cells Results in Agonist Activation of Adenylyl Cyclase and Agonist‐Mediated Selective Down‐Regulation of Gsα

Abstract: Murine neuroblastoma × embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human β2‐adrenoceptor under the control of a β‐actin promoter. Two clones were selected for detailed analysis: D1, which expressed some 12.7 pmol/mg of membrane protein, and L9, which expressed 1.2 pmol/mg of membrane protein of the receptor. Incubation with the β‐adrenoceptor agonist isoprenaline resulted in stimulation of adenylyl cyclase activity in both of the clones, whereas no such activation was observed in wild‐type NCB20 cells. The EC50 for isoprenaline stimulation of adenylyl cyclase activity in membranes of clone D1 (0.8 nM) was significantly lower, however, than in membranes of clone L9 (10.4 nM). Although the maximal adenylyl cyclase stimulation by isoprenaline was similar in both clones, D1 had a higher basal activity. Immunoblotting studies with specific antipeptide antisera directed against various G protein α subunits showed that treatment of the cells with isoprenaline resulted in a 35% reduction in the membrane‐associated levels of Gsα in membranes of clone L9 cells and a 50% reduction in Gsα levels in membranes prepared from clone D1. Isoprenaline treatment had no effect on the levels of Gsα in wild‐type NCB20 cells, and such treatment had no effect on the levels of other G protein α subunits such as Gq/G11 and Gi2 in any of the cell lines investigated. Time course analysis revealed that half‐maximal loss of Gsα in clone D1 was achieved within 1–2 h of addition of agonist. Dose‐response curves to isoprenaline in clone D1 indicated that half‐maximal down‐regulation of Gsα was produced by ∼1 nM agonist. Measurement of Gs mRNA levels in both clones, however, using both reverse transcriptase‐polymerase chain reaction and northern blotting revealed no significant change following treatment with isoprenaline.

Isolation of a putative receptor from Zea mays microsomal membranes that interacts with the G-protein, GPα1

The C‐terminal region of a heterotrimeric G‐protein α‐subunit is known to be one of the principal determinants governing its interaction with its cognate receptor. Use of an oligopeptide corresponding to the fifteen C‐terminal residues of the Arabidopsis Gα‐subunit (GPα1), as an affinity ligand, led to the resolution of a tightly binding 37 kDa membrane polypeptide from detergent solubilised Zea microsomal fraction membranes. An identical polypeptide bound tightly to an affinity matrix containing recombinant GPα1 protein as ligand: binding and release of this 37 kDa protein was dependent on the activation state of GPα1 which was regulated by inclusion or omission of the G‐protein activator AlF− 4. Finally, the isolated 37 kDa protein was labelled with the lectin concanavalin A, indicating it to be glycosylated. These data are consistent with the identity of the 37 kDa membrane polypeptide as a receptor that interacts with the Zea homologue of GPα1.