Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled receptors.

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.

Expression of beta-arrestins and beta-adrenergic receptor kinases in the failing human heart.

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.

Overexpression of beta-arrestin and beta-adrenergic receptor kinase augment desensitization of beta 2-adrenergic receptors.

Receptor-specific or homologous desensitization of beta 2-adrenergic receptors is thought to be effected via phosphorylation of the receptor by the beta-adrenergic receptor kinase (beta ARK), followed by binding of beta-arrestin. We have generated stably transfected Chinese hamster ovary cell lines overexpressing either of the two regulatory proteins and also expressing low or high levels of beta 2-adrenergic receptors (approximately 80 and approximately 600 fmol/mg of membrane protein). In these cells, we studied the process of desensitization induced by the beta-adrenergic receptor agonist isoproterenol.

Phosducin is a protein kinase A-regulated G-protein regulator.

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.

Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium.

Intragenic mutations were isolated that suppressed the dominant-lethal phenotype of the YPT1ile121 mutant gene in a temperature-dependent fashion. Among different amino acid substitutions resulting from single point mutations, two, Ala161----Val (A161V) and Met165----Ile (M165I), restored the function of the YPT1ile121 mutant protein. Mutants expressing the YPT1ile121/val161 allele (ypt1ts) only, grew normally at temperatures up to 30 degrees C but were arrested at 37 degrees C.