%0 Journal Article %1 Sohlemann1995 %A Sohlemann, P. %A Hekman, M. %A Puzicha, M. %A Buchen, C. %A Lohse, M. J. %D 1995 %J Eur J Biochem %K *Arrestins AMP-Dependent Adenylate Animals Antigens/genetics/*metabolism Binding Cell Cyclase/metabolism Cyclic Expression Eye GTP-Binding Gene Humans Kinases Kinases/metabolism Kinetics Line Outer Phosphorylation Protein Proteins/genetics/*metabolism Proteins/genetics/metabolism Receptor Recombinant Rhodopsin/metabolism Rod Segment/metabolism Spodoptera beta-2/chemistry/genetics/*metabolism beta-Adrenergic Proteins/metabolism Adrenergic %N 2 %P 464-72 %T Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled receptors %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7556195 %V 232 %X 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.

@article{Sohlemann1995, abstract = {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.}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Sohlemann, P. and Hekman, M. and Puzicha, M. and Buchen, C. and Lohse, M. J.}, biburl = {https://www.bibsonomy.org/bibtex/27b537bf6cde44158ccbc56dd59a49b24/pharmawuerz}, endnotereftype = {Journal Article}, interhash = {245e5223aa110c01af5ac9adde691a53}, intrahash = {7b537bf6cde44158ccbc56dd59a49b24}, issn = {0014-2956 (Print) 0014-2956 (Linking)}, journal = {Eur J Biochem}, keywords = {*Arrestins AMP-Dependent Adenylate Animals Antigens/genetics/*metabolism Binding Cell Cyclase/metabolism Cyclic Expression Eye GTP-Binding Gene Humans Kinases Kinases/metabolism Kinetics Line Outer Phosphorylation Protein Proteins/genetics/*metabolism Proteins/genetics/metabolism Receptor Recombinant Rhodopsin/metabolism Rod Segment/metabolism Spodoptera beta-2/chemistry/genetics/*metabolism beta-Adrenergic Proteins/metabolism Adrenergic}, month = {Sep 1}, note = {Sohlemann, P Hekman, M Puzicha, M Buchen, C Lohse, M J Research Support, Non-U.S. Gov't Germany European journal of biochemistry / FEBS Eur J Biochem. 1995 Sep 1;232(2):464-72.}, number = 2, pages = {464-72}, shorttitle = {Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled receptors}, timestamp = {2010-12-14T18:22:42.000+0100}, title = {Binding of purified recombinant beta-arrestin to guanine-nucleotide-binding-protein-coupled receptors}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=7556195}, volume = 232, year = 1995 }