Signal transduction by G-protein-coupled receptors is regulated by various mechanisms acting at the receptor level; those studied most thoroughly are from the beta-adrenergic receptor/Gs/adenylyl cyclase system. We report here a regulatory mechanism occurring at the level of the G proteins themselves. A protein with M(r) 33,000 that inhibits Gs-GTPase activity was purified from bovine brain. This protein is very similar or identical to phosducin, a protein previously thought to be specific for retina and pineal gland. Recombinant phosducin inhibited the GTPase activity of several G proteins, and also inhibited Gs-mediated adenylyl cyclase activation. Blockade of its inhibitory effects by protein kinase A suggests that phosducin may be part of a complex regulatory network controlling G-protein-mediated signalling.

The beta-adrenergic receptor system of the failing human heart is markedly desensitized. We have recently postulated that this desensitization may in part be caused by an increase in beta-adrenergic receptor kinase (beta ARK) expression. beta ARK is thought to effect desensitization by acting in concert with an inhibitor protein, called beta-arrestin. Two isoforms have been identified both for beta ARK and for beta-arrestin. In the present study, we have investigated the expression of the individual isoforms of beta-arrestin and of beta ARK in left ventricles from failing and control human hearts. mRNAs for all four proteins, beta-arrestin-1, beta-arrestin-2, beta ARK-1, and beta ARK-2, were identified in human heart. Quantitation by reverse-transcription polymerase chain reactions showed that in heart failure there were no changes of the mRNA levels for beta-arrestin-1 and beta-arrestin-2, a slight (< 50%) increase of the mRNA for beta ARK-2, and a threefold increase for beta ARK-1 mRNA. At the protein level, beta-arrestin-1 was readily detected by Western blotting in human heart. Its absolute values were approximately 350 fmol/mg cytosolic protein, and its expression was not changed in heart failure. beta-Arrestin-2 levels were too low to be detectable using the same methods. beta ARK levels as determined by enzymatic activity were approximately 20 fmol/mg cytosolic protein (beta ARK-1 plus beta ARK-2) and thus almost 20-fold lower than those of beta-arrestin. beta ARK levels were increased approximately twofold in heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)

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beta-arrestin is a cytosolic protein thought to be responsible for uncoupling agonist-activated beta 2-adrenergic receptors from their guanine-nucleotide-binding proteins (G-protein) subsequent to receptor phosphorylation by the beta-adrenergic receptor kinase (beta ARK). In order to investigate this interaction, we generated a recombinant baculovirus for the expression of beta-arrestin in Sf9 insect cells. Apparently homogeneous beta-arrestin preparations were obtained in a one-step purification on heparin-Sepharose. Purified beta-arrestin bound to rhodopsin in a phosphorylation-dependent plus light-dependent manner. Binding to beta 2-adrenergic receptors was investigated using purified receptors reconstituted into lipid vesicles. The accessibility of the reconstituted receptors was determined using the agonist isoproterenol for the ligand-binding site and an antibody binding to an attached myc tag for the C-terminus, the site of receptor phosphorylation. On the basis of these data, the binding of purified beta-arrestin to beta ARK-phosphorylated beta 2-adrenergic receptors was found to occur with a KD of 1.8 nM and with a maximum of 1 beta-arrestin/receptor. beta-arrestin also bound to receptors which had been completely dephosphorylated with acid phosphatase, but the affinity was approximately 30-fold lower. In contrast to regulation by phosphorylation, binding of agonists or antagonists to the receptors had negligible effects on beta-arrestin binding. Finally, beta-arrestin and beta ARK were shown to be capable of producing synergistic inhibition of beta 2-adrenergic-receptor-stimulated adenylyl cyclase activity of cell membranes. These data show that high-affinity stoichiometric binding of beta-arrestin to beta 2-adrenergic receptors occurs in a beta ARK-dependent manner and is sufficient to impair adenylyl cyclase stimulation by the receptors.

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