Synthetic peptides of the hamster beta 2-adrenoceptor as substrates and inhibitors of the beta-adrenoceptor kinase.

1. The beta-adrenoceptor is one of a number of G protein-coupled receptors which have been proposed to contain seven transmembrane alpha-helices. The function of this receptor appears to be regulated by phosphorylation by a specific enzyme, the beta-adrenoceptor kinase. Synthetic peptides which comprise each of the proposed intra- and extracellular domains of the beta 2-adrenoceptor have been tested as potential substrates and inhibitors of the beta-adrenoceptor kinase. 2. Two peptides which encompass the middle and terminal portions of the carboxyl tail of the receptor served as substrates by beta-adrenoceptor kinase. The kinetics of the phosphorylation reaction, however, suggest that these peptides are 10(6)-fold poorer substrate than the agonist occupied receptor. 3. A number of synthetic peptides also served as inhibitors of beta 2-adrenoceptor phosphorylation by beta-adrenoceptor kinase. In particular, a peptide which comprised the first intracellular loop of the beta 2-adrenoceptor (amino acids 56-74) inhibited most effectively with an IC50 of 40 microM. 4. These results suggest that multiple intracellular regions of the beta-receptor may serve as potential sites of interaction with beta-adrenoceptor kinase. Moreover, these regions may serve as potential targets for the development of specific inhibitors of beta-adrenoceptor kinase which could be used to block homologous desensitization.

beta-Adrenergic receptor kinase. Activity of partial agonists for stimulation of adenylate cyclase correlates with ability to promote receptor phosphorylation.

The beta-adrenergic receptor (beta AR) kinase is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor. We have utilized the agonist-dependent nature of this phosphorylation reaction to characterize the ability of partial agonists to interact with the receptor. Partial agonists were tested for their ability to: 1) stimulate adenylate cyclase activity in a three-component reconstituted system, and 2) promote phosphorylation of beta AR by beta AR kinase. There is an excellent correlation between the ability of partial agonists to stimulate adenylate cyclase activity and promote receptor phosphorylation by beta AR kinase (y = 1.02x-0.01, r = 0.996, p less than 0.001). Peptide maps of receptor phosphorylated by beta AR kinase in the presence of full or partial agonists are virtually identical with the partial agonist pattern reduced in intensity. Moreover, kinetic studies of beta AR phosphorylation by beta AR kinase suggest that partial agonists alter the Vmax of the reaction with little, if any, effect on the Km. These results suggest that at steady state partial agonists transform a smaller portion of the receptor pool into the conformationally altered or activated form which serves as the substrate for beta AR kinase, although they do not completely rule out the possibility that a partial conformational change is occurring.

Purification and characterization of the beta-adrenergic receptor kinase.

The beta-adrenergic receptor kinase (beta-ARK) is a recently discovered enzyme which specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta-AR) as well as the light-bleached form of rhodopsin. beta-ARK is present in a wide variety of mammalian tissues. The kinase can be purified from bovine cerebral cortex to greater than 90% homogeneity by sequential chromatography on Ultrogel AcA34, DEAE-Sephacel, CM-Fractogel, and hydroxylapatite. This results in an approximately 20,000-fold purification with an overall recovery of 12%. The purified kinase has an Mr approximately 80,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Several findings indicate that this peptide contains the beta-ARK activity. First, on hydroxylapatite chromatography the enzyme activity coelutes with the Mr approximately 80,000 protein as revealed by Coomassie-Blue staining. Second, under phosphorylating conditions the Mr approximately 80,000 protein is phosphorylated. Finally, the Mr approximately 80,000 protein specifically interacts with reconstituted agonist-occupied beta-AR. Kinetic parameters of the enzyme for beta-AR are Km = 0.25 microM and Vmax = 78 nmol/min/mg whereas for rhodopsin the values are Km = 6 microM and Vmax = 72 nmol/min/mg. The Km value of the enzyme for ATP is approximately 35 microM using either beta-AR or rhodopsin as substrate. Receptor phosphorylation by beta-ARK is effectively inhibited by Zn2+, digitonin and a variety of salts. The availability of purified beta-ARK should greatly facilitate studies of its role in receptor desensitization.

Functional differences in the beta gamma complexes of transducin and the inhibitory guanine nucleotide regulatory protein.

We have examined the mechanism of inhibition of adenylate cyclase using the purified alpha and beta gamma subunits of bovine brain inhibitory guanine nucleotide regulatory protein (Ni) (i.e., alpha i and beta gamma N) and bovine retinal transducin (alpha T and beta gamma T) in reconstituted phospholipid vesicle systems. The addition of beta gamma N or beta gamma T to lipid vesicles containing the pure stimulatory guanine nucleotide regulatory protein (Ns) from human erythrocytes as well as a resolved preparation of the catalytic moiety (C) of bovine caudate adenylate cyclase results in significant inhibition of guanine nucleotide stimulated cyclase activity (80-90%). The inhibition by these beta gamma subunit complexes appears to fully account for the inhibitory effects observed with holo-Ni or holotransducin. A variety of structure-function comparisons of the beta gamma N and beta gamma T complexes were performed in order to further probe the molecular mechanisms involved in the inhibitory pathway. Whereas the beta subunits of beta gamma N and beta gamma T appear to be very similar, if not identical, on the basis of comparisons of their gel electrophoretic mobility and immunological cross-reactivity, clear differences exist in the apparent structures of gamma N and gamma T. Interestingly, functional differences are observed in the effectiveness of these two beta gamma complexes to inhibit adenylate cyclase activity. Specifically, while both beta gamma N and beta gamma T are capable of effecting significant levels of inhibition of the guanine nucleotide stimulated activities, the beta gamma N complex is consistently more potent than beta gamma T in inhibiting these activities.(ABSTRACT TRUNCATED AT 250 WORDS)

The mammalian beta-adrenergic receptor: structural and functional characterization of the carbohydrate moiety.

Mammalian beta-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of Mr approximately 64,000. Treatment of purified [125I]-labeled hamster lung beta-adrenergic receptor with alpha-mannosidase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [125I]-labeled receptor with endoglycosidase F results initially in the formation of a Mr approximately 57,000 peptide which is further converted to Mr approximately 49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [125I]-labeled receptor reveals the presence of two complex type carbohydrate chains (approximately 10% of which are fucosylated) on approximately 45% of the receptors. The remaining approximately 55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of beta-adrenergic agonists and antagonists. Moreover, the ability of control, alpha-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, alpha-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleotide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the beta-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.

Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system.

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.

A role for Ni in the hormonal stimulation of adenylate cyclase.

The best understood system for transduction of extracellular messages into intracellular signals is the hormone receptor-coupled adenylate cyclase. In such systems receptors are functionally coupled to the enzyme by two special proteins, termed the stimulatory and inhibitory guanine nucleotide regulatory proteins (Ns and Ni, respectively). These proteins, thought to mediate, respectively, stimulatory and inhibitory influences on the adenylate cyclase, are members of a larger class of heterotrimeric guanine nucleotide regulatory proteins involved in membrane signal transduction. We have studied the interactions of the various components of the adenylate cyclase system by co-reconstituting pure beta-adrenergic receptors, pure Ns and Ni, and functionally resolved preparations of the catalyst in phospholipid vesicles. In the absence of Ni, beta-adrenergic receptor/Ns-mediated catecholamine stimulation of the enzyme is relatively modest (approximately 1.3-fold). Surprisingly, however, when Ni is also present, stimulation increases dramatically (up to 7-8-fold) because of a greater suppression of basal relative to agonist-stimulated enzyme activity. Thus, Ni may actually be required for maximal agonist stimulation as well as for inhibition of the adenylate cyclase.

Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems.

The adenylate cyclase coupled inhibitory nucleotide regulatory protein (Ni) and the bovine retinal nucleotide regulatory protein transducin (T) appear to share some common functional properties since their GTPase activity is stimulated to similar extents by the retinal photoreceptor rhodopsin. In the present work, we sought to assess whether these functional similarities might extend to their interaction with adenylate cyclase. This necessitated the development of reconstitution systems in which guanine nucleotide regulatory protein mediated inhibition of adenylate cyclase activity could be demonstrated and characterized in a lipid milieu. In the absence of the pure human erythrocyte stimulatory nucleotide regulatory protein (Ns), the insertion into phospholipid vesicles of either pure Ni from human erythrocytes or pure bovine T with the resolved catalytic moiety of bovine caudate adenylate cyclase (C) does not establish GppNHp inhibition of either Mg2+- or forskolin-stimulated adenylate cyclase. However, the coinsertion into lipid vesicles of either Ni or T with Ns and resolved C results in an inhibition of Ns(GppNHp) stimulatable C activity. As is the case in intact membranes, the reconstituted inhibition of the Ns-stimulated C activity extends into the steady-state phase of time courses of activity. This inhibition is highly sensitive to the MgCl2 concentration. At 2 mM MgCl2, the inhibition is greater than 80% while at 50 mM MgCl2 it is only approximately 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorylation of the mammalian beta-adrenergic receptor by cyclic AMP-dependent protein kinase. Regulation of the rate of receptor phosphorylation and dephosphorylation by agonist occupancy and effects on coupling of the receptor to the stimulatory guanine nucleotide regulatory protein.

In some systems, such as the turkey erythrocyte, agonist-promoted phosphorylation of the beta-adrenergic receptor appears to be associated with desensitization of the adenylate cyclase system. This process can be partially mimicked by cyclic AMP analogs. Accordingly, we have investigated the phosphorylation of the pure mammalian beta-adrenergic receptor by the pure catalytic subunit of the cyclic AMP-dependent protein kinase. The beta-adrenergic receptor, purified from hamster lung to apparent homogeneity, contains a single polypeptide of Mr approximately 64,000. The receptor can be phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase (approximately 2 mol of phosphate (on serine residues) per mol). Isoproterenol, a beta-agonist, promoted a 2-3-fold increase in the rate of receptor phosphorylation which was blocked by the beta-antagonists propranolol and alprenolol. High performance liquid chromatographic tryptic peptide mapping reveals two major phosphorylation sites. Phosphorylated receptor can be completely dephosphorylated by a high molecular weight phosphoprotein phosphatase. The rate of receptor dephosphorylation is enhanced 2-3-fold by isoproterenol and this effect is blocked by alprenolol. The functional significance of receptor phosphorylation was examined using ligand binding and reconstitution techniques. While the binding of isoproterenol and alprenolol to the receptor was unaffected by phosphorylation, the ability of the receptor to interact with the stimulatory guanine nucleotide regulatory protein, as assessed by isoproterenol-promoted GTPase activity, was decreased 24 +/- 1% (mean +/- S.E., p less than 0.001, n = 17). The quantitative extent of receptor phosphorylation and functional impairment are virtually identical to those previously observed when intact turkey erythrocytes were incubated with cyclic AMP. These data provide a direct demonstration of regulation of the function of the isolated beta-adrenergic receptor by cyclic AMP-dependent protein kinase.

Specificity of the functional interactions of the beta-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles.

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung beta-adrenergic receptor (beta AR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (Ns) and inhibitory (Ni) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of Ns with beta AR in lipid vesicles increases the extent of binding of [35S] GTP gamma S to Ns and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of Pi released/min/mol of Ns-guanine nucleotide complexes). Enhancement of this turnover rate requires the beta-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTP gamma S binding to Ns. The co-insertion of the beta AR with Ni or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTP gamma S binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with beta AR is Ns much greater than Ni greater than T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T approximately equal to Ni much greater than Ns. Nonetheless the fold stimulation of GTPase activity and [35S]GTP gamma S binding in T, induced by light-activated rhodopsin, is significantly greater than the "fold" stimulation of these activities in Ni. This reflects the greater intrinsic ability of Ni to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S] GTP gamma S) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both Ni and T are similar to those obtained for isoproterenol-stimulated activity in Ns. This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.