Abstract
Co-expression of the alpha(1b)-adrenoreceptor and Galpha(11) in cells derived from a Galpha(q)/Galpha(11) knock-out mouse allows agonist-mediated elevation of intracellular Ca(2+) levels that is transduced by beta/gamma released from the G protein alpha subunit. Mutation of Tyr(356) of Galpha(11) to Phe, within a receptor contact domain, had little effect on function but this was reduced greatly by alteration to Ser and virtually eliminated by conversion to Asp. This pattern was replicated following incorporation of each form of Galpha(11) into fusion proteins with the alpha(1b)-adrenoreceptor. Following a [(35)S]guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding assay, immunoprecipitation of the wild type alpha(1b)-adrenoreceptor-Galpha(11) fusion protein indicated that the agonist phenylephrine stimulated guanine nucleotide exchange on Galpha(11) more than 30-fold. Information transfer by agonist was controlled in residue 356 Galpha(11) mutants with rank order Tyr > Phe > Trp > Ile > Ala = Gln = Arg > Ser > Asp, although these alterations did not alter the binding affinity of either phenylephrine or an antagonist ligand. Mutation of a beta/gamma contact interface in the alpha(1b)-adrenoreceptor-Tyr(356) Galpha(11) fusion protein did not alter ligand binding affinity but did reduce greatly beta/gamma binding and phenylephrine stimulation of [(35)S]GTPgammaS binding. It also prevented agonist elevation of intracellular Ca(2+) levels, as did a mutation in Galpha(11) that prevents G protein subunit dissociation. These results indicate that a bulky aromatic group is required four amino acids from the C terminus of Galpha(11) to maximize information transfer from an agonist-occupied receptor and disprove the hypothesis that tyrosine phosphorylation of this residue is required for G protein activation (Umemori, H., Inoue, T., Kume, S., Sekiyama, N., Nagao, M., Itoh, H., Nakanishi, S., Mikoshiba, K., and Yamamoto, T. (1997) Science 276, 1878-1881). This is distinct from Galpha(i1), where hydrophobicity of the amino acid is the key determinant at this location. They also further demonstrate a key role for the beta/gamma complex in enhancing receptor to G protein alpha subunit information transfer.
Highlights
Protein and its subsequent replacement by GTP [1]
Previous studies have suggested that this Tyr can become phosphorylated in response to G protein-coupled receptors (GPCRs) activation and that this may be a key event in activation of the G protein [13]
The guanine nucleotide binding site and GTPase machinery are defined by the G protein ␣ subunit, the /␥ complex plays a key role, with growing evidence for a direct role of the ␥ subunit in contacting the receptor to enhance guanine nucleotide exchange [15, 16]
Summary
It prevented agonist elevation of intracellular Ca2؉ levels, as did a mutation in G␣11 that prevents G protein subunit dissociation These results indicate that a bulky aromatic group is required four amino acids from the C terminus of G␣11 to maximize information transfer from an agonist-occupied receptor and disprove the hypothesis that tyrosine phosphorylation of this residue is required for G protein activation We have recently employed an immunoprecipitation strategy in concert with a [35S]GTP␥S binding assay to monitor directly GPCR and agonist-induced guanine nucleotide exchange on G␣11 [14] We extend this approach to analyze the effects on information transfer between the ␣1b-adrenoreceptor and forms of G␣11 in which Tyr356 was altered to a range of amino acids. Overexpression of /␥ with a fusion protein between the ␣1b-adrenoreceptor and the mutant form of G␣11 with reduced /␥ binding affinity enhances the effectiveness of agonist-stimulated guanine nucleotide exchange
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