An intact N terminus of the gamma subunit is required for the Gbetagamma stimulation of rhodopsin phosphorylation by human beta-adrenergic receptor kinase-1 but not for kinase binding.

Cleavage after lysine 32 in the Ggamma2 subtype and after lysine 36 in the Ggamma3 subtype of purified mixed brain Gbetagamma by endoproteinase Lys-C blocks Gbetagamma-mediated stimulation of phosphorylation of rhodopsin in urea-extracted rod outer segments by recombinant human beta-adrenergic receptor kinase (hbetaARK1) holoenzyme while hbetaARK1 binding to rod outer segments is partially affected. This treatment does not attenuate the binding of the treated Gbetagamma to C-terminal fragments of hbetaARK1 containing the pleckstrin homology domain. Lys-C proteolysis also does not alter the association of the Gbetagamma with phospholipids, its ability to support pertussis toxin-catalyzed Galphao/Galphai ADP-ribosylation, or its ability to inhibit forskolin-stimulated platelet adenylate cyclase. The Gbeta subunit remains noncovalently associated with the cleaved Ggamma fragments. Thus, in addition to recruiting hbetaARK1 to its receptor substrate, Ggamma contributes secondary and/or tertiary structural features to activate the kinase.

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. 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)

Role of extracellular disulfide-bonded cysteines in the ligand binding function of the beta 2-adrenergic receptor.

Evidence is presented for a role of disulfide bridging in forming the ligand binding site of the beta 2-adrenergic receptor (beta AR). The presence of disulfide bonds at the ligand binding site is indicated by "competitive" inhibition by dithiothreitol (DTT) in radioligand binding assays, by specific protection by beta-adrenergic ligands of these effects, and by the requirement of disulfide reduction for limit proteolysis of affinity ligand labeled receptor. The kinetics of binding inhibition by DTT suggest at least two pairs of disulfide-bonded cysteines essential for normal binding. Through site-directed mutagenesis, we indeed were able to identify four cysteines which are critical for normal ligand binding affinities and for the proper expression of functional beta AR at the cell surface. Unexpectedly, the four cysteines required for normal ligand binding are not those located within the hydrophobic transmembrane domains of the receptor (where ligand binding is presumed to occur) but lie in the extracellular hydrophilic loops connecting these transmembrane segments. These findings indicate that, in addition to the well-documented involvement of the membrane-spanning domains of the receptor in ligand binding, there is an important and previously unsuspected role of the hydrophilic extracellular domains in forming the ligand binding site.

Substitution of an extracellular cysteine in the beta 2-adrenergic receptor enhances agonist-promoted phosphorylation and receptor desensitization.

We constructed and expressed in a permanent cell line a beta 2-adrenergic receptor with a valine substitution for cysteine 184 of the second putative extracellular loop. The mutant receptor was partially uncoupled from adenylyl cyclase with impaired ability to form the high affinity agonist-receptor-G protein complex, yet displayed more rapid and extensive agonist-induced desensitization. The enhanced desensitization was accompanied by increased agonist promoted, but not cAMP promoted, receptor phosphorylation in intact cells. Thus, not only is impaired desensitization associated with decreased phosphorylation, as we have shown with several mutant beta 2-adrenergic receptors recently, but enhanced desensitization is accompanied by increased agonist promoted receptor phosphorylation. In the case of this cysteine mutant, this may be due to the greater accessibility of the uncoupled receptor for phosphorylation by the beta-adrenergic receptor kinase.

Beta-adrenergic receptor kinase: primary structure delineates a multigene family.

The beta-adrenergic receptor kinase (beta-ARK), which specifically phosphorylates only the agonist-occupied form of the beta-adrenergic and closely related receptors, appears to be important in mediating rapid agonist-specific (homologous) desensitization. The structure of this enzyme was elucidated by isolating clones from a bovine brain complementary DNA library through the use of oligonucleotide probes derived from partial amino acid sequence. The beta-ARK cDNA codes for a protein of 689 amino acids (79.7 kilodaltons) with a protein kinase catalytic domain that bears greatest sequence similarity to protein kinase C and the cyclic adenosine monophosphate (cyclic AMP)--dependent protein kinase. When this clone was inserted into a mammalian expression vector and transfected into COS-7 cells, a protein that specifically phosphorylated the agonist-occupied form of the beta 2-adrenergic receptor and phosphorylated, much more weakly, the light-bleached form of rhodopsin was expressed. RNA blot analysis revealed a messenger RNA of four kilobases with highest amounts in brain and spleen. Genomic DNA blot analysis also suggests that beta-ARK may be the first sequenced member of a multigene family of receptor kinases.

Calculating receptor number from binding experiments using same compound as radioligand and competitor.

Saturation experiments using increasing concentrations of radioligand are commonly used to determine receptor number and affinity, but this protocol is not feasible in all situations. Alternatively, competitive binding experiments are often performed in which binding of a single concentration of radioligand is completed for by multiple concentrations of the same unlabelled ligand, but the analysis of such data has been difficult. Antonio DeBlasi and colleagues present here a simple method for calculating receptor number and affinity from competitive binding data. This experimental protocol is useful when a single class of binding site is present, but is often not able to detect the presence of two classes of site.

Expression of a human cDNA encoding the beta 2-adrenergic receptor in Chinese hamster fibroblasts (CHW): functionality and regulation of the expressed receptors.

A human beta-adrenergic receptor cDNA was transfected and expressed in transformed Chinese hamster fibroblasts (CHW). The expressed receptor exhibited a typical beta 2-adrenergic selectivity for agonists and antagonists as assessed by radioligand binding and adenylate cyclase activation. Guanine nucleotide-sensitive high affinity binding of the agonist, isoproterenol, indicated effective coupling of the expressed receptor to a guanine nucleotide-regulatory protein. The level of expression of beta 2-AR in various cell clones varied over 200-fold and was positively correlated with the levels of beta 2-AR mRNA. In cells expressing between 0.04 and 3.0 pmol of beta 2-AR/mg of membrane protein, the efficacy of isoproterenol for stimulating adenylate cyclase increased with increasing numbers of expressed receptors but reached a plateau and started to decrease in clones with higher beta 2-AR density (3.0-8.0 pmol/mg of membrane protein). Preincubation of beta 2-AR-expressing cells with isoproterenol for 15 min led to significant reduction in the level of isoproterenol-sensitive adenylate cyclase activity. This agonist-induced desensitization was also accompanied by phosphorylation of the beta 2-AR. These data indicate that the expressed human beta 2-AR displays typical functional characteristics of adenylate cyclase-coupled receptors including agonist-induced desensitization. Moreover, the availability of this series of cellular clones, which differ markedly in their density of beta 2-AR, provides a unique set of biological reagents for future studies of beta 2-AR function and regulation.

Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of alpha 1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells.

Continuous exposure of DDT1 MF-2 smooth muscle cells to 10-100 microM norepinephrine results in a dramatic attenuation of the ability of norepinephrine to stimulate inositol phospholipid hydrolysis via alpha 1-adrenergic receptors (alpha 1-AR). In addition to the functional desensitization, norepinephrine exposure also reduces the number of accessible cell surface alpha 1-AR as assayed by [3H]prazosin binding at 4 degrees C. Desensitization of the cells with norepinephrine results in an increase in the phosphorylation of the Mr 80,000 alpha 1-AR ligand binding peptide (2.4 +/- 0.2 mol of 32P per mol of alpha 1-AR; n = 5) when compared to control cells (1.1 +/- 0.1 mol of 32P per mol of alpha 1-AR; n = 5). The time courses of these three processes are all comparable being half-maximal within 1-2 min. These norepinephrine-promoted effects can be prevented by the alpha 1-AR receptor antagonist phentolamine indicating that they are mediated via the alpha 1-AR. Treatment of cells with the vasoactive peptide bradykinin (10 microM) induces desensitization of alpha 1-AR function similar to that induced by tumor-promoting phorbol ester treatment (Leeb-Lundberg, L. M. F., Cotecchia, S., Lomasney, J. W., DeBernardis, J. F., Lefkowitz, R. J., and Caron, M. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5651-5655). Both treatments also result in phosphorylation of the alpha 1-AR, with stoichiometries of 1.7 +/- 0.1 (bradykinin; n = 5) and 3.6 +/- 0.1 (PMA; n = 5) mol of 32P/mol of alpha 1-AR. However, neither phorbol esters nor bradykinin reduce the number of accessible cell surface alpha 1-AR. Similar phosphopeptide maps are obtained from tryptic phosphopeptides generated from phosphorylated alpha 1-AR derived from cells treated with norepinephrine, phorbol 12-myristate 13-acetate, and bradykinin. Phosphoamino acid analysis reveals that the various agents induce phosphorylation on both serine and threonine residues. Thus, phosphorylation of receptors linked to the inositol phospholipid/Ca2+ signaling pathway may represent an important mechanism of regulation of receptor responsiveness.

In vivo regulation of beta-adrenergic receptors on human mononuclear leukocytes: assessment of receptor number, location, and function after posture change, exercise, and isoproterenol infusion.

We studied the regulation of beta-adrenergic receptors in human mononuclear leukocytes (MNL). Total receptor number was determined as specific binding at 4 C of [3H] dihydroalprenolol or [125I]iodopindolol, and redistributed receptors were defined as those binding sites to which the hydrophilic antagonist CGP-12177 did not have access. Receptor function was assessed as cAMP accumulation stimulated by isoproterenol. In in vitro experiments, high concentrations of isoproterenol desensitized receptor function and promoted redistribution of about 80% of the receptors away from the cell surface. However, three in vivo protocols (upright posture for 3 h, moderate exercise, and infusion of isoproterenol for 30 min) redistributed few beta-adrenergic receptors on MNL. The 30-min isoproterenol infusion did not alter later cAMP accumulation, but posture change and exercise increased isoproterenol-stimulated cAMP accumulation in intact MNL. Infusion of isoproterenol for 120 min redistributed 9 +/- 2% (+/- SEM) of the receptors and decreased isoproterenol-stimulated cAMP accumulation by 19 +/- 6%. Isoproterenol-stimulated adenylate cyclase activity in membranes isolated from MNL previously was found to be decreased with upright posture, and we confirmed these findings in assays that did not include exogenous GTP, but instead relied upon guanine nucleotides retained in the membrane preparation. However, when excess GTP was included, isoproterenol-stimulated adenylate cyclase activity in MNL membranes was not altered by posture change. We conclude that substantial receptor redistribution of beta-receptors on MNL does not readily occur in physiological situations.

Desensitization and redistribution of beta-adrenergic receptors on human mononuclear leukocytes.

We have used intact human mononuclear leukocytes (MNL) to examine desensitization of beta-adrenergic receptors in normal mammalian cells. MNL were prepared and radioligand binding experiments were performed at 4 degrees C. At this temperature the ligand [125I]iodocyanopindolol ([125I]ICYP) identified the same number of receptors as at 37 degrees C, and the agonist isoproterenol competed for this binding with high affinity (dissociation constant, Ki = 20 nM). At 37 degrees C, results were similar when the binding incubation was terminated after 1 min, but the apparent affinity of the receptors for isoproterenol was several 100-fold lower when the incubation was allowed to reach steady state. In desensitized MNL (prepared by incubating whole blood with 10 microM isoproterenol at 37 degrees C for 10 min, and then isolating and washing the MNL at 4 degrees C), isoproterenol-stimulated cAMP accumulation was reduced 63 +/- 4%. After desensitization, the total number of beta-receptors was unchanged, but isoproterenol and the hydrophilic antagonist CGP-12177 were able to compete with [125I]ICYP for binding to only 18 +/- 6% of these sites. Direct binding with [3H]CGP-12177 yielded similar results. These results demonstrate that isoproterenol promotes a rapid desensitization of beta-adrenergic receptors on MNL and a concomitant redistribution of receptors into a cellular compartment to which some ligands (including catecholamines) have restricted access. The findings demonstrate that redistribution of beta-receptors may be a mechanism mediating desensitization to catecholamines in normal mammalian cells.