Somatostatin induces translocation of the beta-adrenergic receptor kinase and desensitizes somatostatin receptors in S49 lymphoma cells

The beta-adrenergic receptor kinase is a cytosolic enzyme that specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta AR). Beta AR kinase appears to be translocated from the cytosol to the plasma membrane when kin- S49 lymphoma cells are incubated with either beta-adrenergic agonists or prostaglandin E1, both of which act through receptors which stimulate adenylate cyclase. We report here that brief (approximately 20 min) exposure of wild type S49 lymphoma cells to somatostatin (which inhibits adenylate cyclase) promotes the translocation of beta AR kinase to an extent comparable to that observed in the presence of the beta agonist isoproterenol or prostaglandin E1. Beta AR kinase activity can be measured using either beta AR or rhodopsin, the retinal receptor for light, as a substrate. The translocation process triggered by somatostatin is rapid, reversible, and is associated with somatostatin receptor desensitization. The latter is apparent as an attenuation of the inhibition by somatostatin of forskolin-stimulated adenylate cyclase activity in membranes of S49 cells preincubated in the presence of the peptide. These results strongly suggest that beta AR kinase is able to phosphorylate and desensitize both stimulatory and inhibitory adenylate cyclase-coupled receptors, thus emerging as a general kinase that regulates the function of different receptors in an agonist-specific fashion.     

Receptor-specific desensitization with purified proteins. Kinase dependence and receptor specificity of beta-arrestin and arrestin in the beta 2-adrenergic receptor and rhodopsin systems.

Homologous desensitization of beta-adrenergic receptors, as well as adaptation of rhodopsin, are thought to be triggered by specific phosphorylation of the receptor proteins. However, phosphorylation alone seems insufficient to inhibit receptor function, and it has been proposed that the inhibition is mediated, following receptor phosphorylation, by the additional proteins beta-arrestin in the case of beta-adrenergic receptors and arrestin in the case of rhodopsin. In order to test this hypothesis with isolated proteins, beta-arrestin and arrestin were produced by transient overexpression of their cDNAs in COS7 cells and purified to apparent homogeneity. Their functional effects were assessed in reconstituted receptor/G protein systems using either beta 2-adrenergic receptors with Gs or rhodopsin with Gt. Prior to the assays, beta 2-receptors and rhodopsin were phosphorylated by their specific kinases beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, respectively. beta-Arrestin was a potent inhibitor of the function of beta ARK-phosphorylated beta 2-receptors. Half-maximal inhibition occurred at a beta-arrestin:beta 2-receptor stoichiometry of about 1:1. More than 100-fold higher concentrations of arrestin were required to inhibit beta 2-receptor function. Conversely, arrestin caused half-maximal inhibition of the function of rhodopsin kinase-phosphorylated rhodopsin when present in concentrations about equal to those of rhodopsin, whereas beta-arrestin at 100-fold higher concentrations had little inhibitory effect. The potency of beta-arrestin in inhibiting beta 2-receptor function was increased over 10-fold following phosphorylation of the receptors by beta ARK, but was not affected by receptor phosphorylation using protein kinase A. This suggests that beta-arrestin plays a role in beta ARK-mediated homologous, but not in protein kinase A-mediated heterologous desensitization of beta-adrenergic receptors. It is concluded that even though arrestin and beta-arrestin are similar proteins, they display marked specificity for their respective receptors and that phosphorylation of the receptors by the receptor-specific kinases serves to permit the inhibitory effects of the "arresting" proteins by allowing them to bind to the receptors and thereby inhibit their signaling properties. Furthermore, it is shown that this mechanism of receptor inhibition can be reproduced with isolated purified proteins.     

Fluorescent localization of the beta-adrenergic receptor on DDT-1 cells. Down-regulation by adrenergic agonists.

Continuous incubation of cultured cells with beta-adrenergic agonists results in the desensitization of adrenergic responsiveness accompanied by the down-regulation of cell surface beta-adrenergic receptors (beta AR). Previous studies have relied on measurements of ligand binding activity for the detection of the beta AR in the cell. In the present study, we have raised a monoclonal antibody to a synthetic peptide corresponding to amino acid numbers 226-239 of the hamster beta 2AR. This antibody was used to localize the beta AR in hamster smooth-muscle DDT-1 cells by immunofluorescence, without regard for the ability of the receptor to bind ligands. The beta AR was found to be localized primarily at the plasma membrane of these cells, with a nonhomogeneous pattern of distribution. A rapid loss of beta AR-specific immunofluorescence, which paralleled receptor down-regulation as measured by ligand-binding activity, was seen with beta-adrenergic agonists, but not with antagonists. In addition, a transient increase in fluorescence was observed after short times of exposure of the cells to agonists. This fluorescence increase may reflect a ligand-induced conformational change in the receptor.     

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.     

G-protein-coupled receptor kinases.

Rhodopsin kinase and the beta-adrenergic receptor kinase (beta ARK) catalyse the phosphorylation of the activated forms of the G-protein-coupled receptors, rhodopsin and the beta 2-adrenergic receptor (beta 2AR), respectively. The interaction between receptor and kinase is independent of second messengers and appears to involve a multipoint attachment of kinase and substrate with the specificity being restricted by both the primary amino acid sequence and conformation of the substrate. Kinetic, functional and sequence information reveals that rhodopsin kinase and beta ARK are closely related, suggesting they may be members of a family of G-protein-coupled receptor kinases.     

Cross-talk between tyrosine kinase and G-protein-linked receptors. Phosphorylation of beta 2-adrenergic receptors in response to insulin.

Protein kinases play a pivotal role in the propagation and modulation of transmembrane signaling pathways. Two major classes of receptors, G-protein-linked and tyrosine kinase receptors not only propagate signals but also are substrates for phosphorylation in response to stimulation by agonist ligands. Insulin (operating via tyrosine kinase receptors) and catecholamines (operating by G-protein-linked receptors) are counterregulatory with respect to lipid and carbohydrate metabolism. How, on a cellular level, these two distinct classes of receptors may cross-regulate each other remains controversial. In the present work we identify a novel cross-talk between members of two distinct classes of receptors, tyrosine kinase (insulin) and G-protein-linked (beta-adrenergic) receptors. Treatment of DDT1 MF-2 hamster vas deferens smooth muscle cells with insulin promoted a marked attenuation (desensitization) of beta-adrenergic receptor-mediated activation of adenylylcyclase. Measured by immune precipitation of beta 2-adrenergic receptors from cells metabolically labeled with [32P]orthophosphate, the basal state of receptor phosphorylation was increased 2-fold by insulin. Phosphoamino acid analysis revealed that for insulin-stimulated cells, the beta 2-adrenergic receptors showed increased phosphorylation on tyrosyl and decreased phosphorylation on threonyl residues. Phosphorylation of the beta-adrenergic receptor was rapid and peaked at 30 min following stimulation of cells by insulin. beta-Adrenergic receptor phosphorylation and attenuation of catecholamine-sensitive adenylylcyclase provide a biochemical basis for the counterregulatory effects of insulin upon catecholamine action.     

Identification of a Gs activator region of the beta 2-adrenergic receptor that is autoregulated via protein kinase A-dependent phosphorylation.

We have localized a G protein activator region of the human beta 2-adrenergic receptor to region beta III-2 (from Arg259 to Lys273). The synthetic beta III-2, corresponding to the C-terminal end of the third cytoplasmic loop, activates Gs at nanomolar concentrations and weakly activates Gi. beta III-2 activates adenylyl cyclase at nanomolar concentrations in wild-type S49 lymphoma membranes, but not in membranes of unc mutant S49 cells, in which Gs is uncoupled from beta-adrenergic stimulation. Phosphorylation of beta III-2 by cAMP-dependent protein kinase A, which is involved in the desensitization of the beta-adrenergic receptor from Gs, drastically reduces the effect of beta III-2 on Gs while potentiating its action on Gi, resulting in a total loss of adenylyl cyclase-stimulating activity. These findings indicate that this receptor sequence is a multipotential G protein activator whose G protein specificity is regulated by protein kinase A.     

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.     

A novel catecholamine-activated adenosine cyclic 3',5'-phosphate independent pathway for beta-adrenergic receptor phosphorylation in wild-type and mutant S49 lymphoma cells: mechanism of homologous desensitization of adenylate cyclase

Virtually all known biological actions stimulated by beta-adrenergic and other adenylate cyclase coupled receptors are mediated by cAMP-dependent protein kinase. Nonetheless, "homologous" or beta-adrenergic agonist-specific desensitization does not require cAMP. Since beta-adrenergic receptor phosphorylation may be involved in desensitization, we studied agonist-promoted receptor phosphorylation during homologous desensitization in wild-type S49 lymphoma cells (WT) and two mutants defective in the cAMP-dependent pathway of beta-agonist-stimulated protein phosphorylation (cyc- cannot generate cAMP in response to beta-adrenergic agonists; kin- lacks cAMP-dependent kinase). All three cell types demonstrate rapid, beta-adrenergic agonist-promoted, stoichiometric phosphorylation of the receptor which is clearly not cAMP mediated. The amino acid residue phosphorylated is solely serine. These data demonstrate, for the first time, that catecholamines can promote phosphorylation of a cellular protein (the beta-adrenergic receptor) via a cAMP-independent pathway. Moreover, the ability of cells with mutations in the adenylate cyclase-cAMP-dependent protein kinase pathway to both homologously desensitize and phosphorylate the beta-adrenergic receptors provides very strong support for the notion that receptor phosphorylation may indeed be central to the molecular mechanism of desensitization.     

Activation by G protein beta gamma subunits of agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin.

We have partially purified a protein kinase that phosphorylates muscarinic receptors (mAChR) in the presence of agonists and have shown that the phosphorylation is stimulated by the beta gamma subunits of the GTP binding protein Go (Haga, K., and Haga, T. (1990) FEBS Lett. 268, 43-47). We report here that rhodopsin is also phosphorylated in a light-dependent manner by the same kinase preparation and that beta gamma subunits derived from Gs, Gi, and Go stimulate the phosphorylation of both rhodopsin and mAChRs. The rhodopsin- and mAChR-phosphorylating activities were eluted in the same fractions using a purification procedure that is essentially the same as that used for the purification of beta-adrenergic receptor kinase (Benovic, J.L., Strasser, R.H., Caron, M.G., and Lefkowitz, R.J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2797-2801) and were inhibited by low concentrations of heparin, an inhibitor of beta-adrenergic receptor kinase, (IC50 = 15 nM), suggesting that both mAChR and rhodopsin are phosphorylated by the same or very similar kinase(s) belonging to the beta-adrenergic receptor kinase family. G protein beta gamma subunits increased the Vmax of the phosphorylation of rhodopsin 12-fold. Kinetic data were consistent with the assumptions that the protein kinase (mAChR kinase) binds rhodopsin and beta gamma subunits in a random order and that the reaction rate is proportional to concentration of the ternary complex. By contrast, the light-dependent phosphorylation of rhodopsin by the rhodopsin kinase was not stimulated by the beta gamma subunits. These results indicate that beta gamma subunits may interact with and activate the mAChR kinase but not rhodopsin kinase and suggest that the beta gamma subunit of G proteins may take part in the desensitization of G protein-linked receptors.     

Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1. A role in receptor sequestration

Agonist-dependent desensitization of the beta-adrenergic receptor requires translocation and activation of the beta-adrenergic receptor kinase1 by liberated Gbetagamma subunits. Subsequent internalization of agonist-occupied receptors occurs as a result of the binding of beta-arrestin to the phosphorylated receptor followed by interaction with the AP2 adaptor and clathrin proteins. Receptor internalization is known to require D-3 phosphoinositides that are generated by the action of phosphoinositide 3-kinase. Phosphoinositide 3-kinases form a family of lipid kinases that couple signals via receptor tyrosine kinases and G-protein-coupled receptors. The molecular mechanism by which phosphoinositide 3-kinase acts to promote beta-adrenergic receptor internalization is not well understood. In the present investigation we demonstrate a novel finding that beta-adrenergic receptor kinase 1 and phosphoinositide 3-kinase form a cytosolic complex, which leads to beta-adrenergic receptor kinase 1-mediated translocation of phosphoinositide 3-kinase to the membrane in an agonist-dependent manner. Furthermore, agonist-induced translocation of phosphoinositide 3-kinase results in rapid interaction with the receptor, which is of functional importance, since inhibition of phosphoinositide 3-kinase activity attenuates beta-adrenergic receptor sequestration. Therefore, agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane is an important step in the process of receptor sequestration and links phosphoinositide 3-kinase to G-protein-coupled receptor activation and sequestration.     

Altered phosphorylation and desensitization patterns of a human beta 2-adrenergic receptor lacking the palmitoylated Cys341.

Exposure of beta 2-adrenergic receptors to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase, associated with an increased phosphorylation of the receptor. Agonist-promoted phosphorylation of the beta 2-adrenergic receptor (beta 2AR) by protein kinase A and the beta-adrenergic receptor kinase (beta ARK) is believed to promote a functional uncoupling of the receptor from the guanyl nucleotide regulatory protein Gs. More recently, palmitoylation of Cys341 of the receptor has also been proposed to play an important role in the coupling of the beta 2-adrenergic receptor to Gs. Here we report that substitution of the palmitoylated cysteine by a glycine (Gly341 beta 2 AR) using site directed mutagenesis leads to a receptor being highly phosphorylated and largely uncoupled from Gs. In Chinese hamster fibroblasts (CHW), stably transfected with the human receptor cDNAs, the basal phosphorylation level of Gly341 beta 2AR was found to be approximately 4 times that of the wild type receptor. This elevated phosphorylation level was accompanied by a depressed ability of the receptor to stimulate the adenylyl cyclase and to form a guanyl nucleotide-sensitive high affinity state for agonists. Moreover, exposure of this unpalmitoylated receptor to an agonist did not promote any further phosphorylation or uncoupling. A modest desensitization of the receptor-stimulated adenylyl cyclase response was observed but resulted from the agonist-induced sequestration of the unpalmitoylated receptor and could be blocked by concanavalin A. This contrasts with the agonist-promoted phosphorylation and uncoupling of the wild type receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

The palmitoylation state of the beta(2)-adrenergic receptor regulates the synergistic action of cyclic AMP-dependent protein kinase and beta-adrenergic receptor kinase involved in its phosphorylation and desensitization

Although palmitoylation of the beta(2)-adrenergic receptor (beta(2)AR), as well as its phosphorylation by the cyclic AMP-dependant protein kinase (PKA) and the beta-adrenergic receptor kinase (beta ARK), are known to play important roles in agonist-promoted desensitization, their relative contribution and mutual regulatory influences are still poorly understood. In this study, we investigated the role that the carboxyl tail PKA site (Ser(345,346)) of the beta(2)AR plays in its rapid agonist-promoted phosphorylation and desensitization. Mutation of this site (Ala(345,346)beta(2)AR) significantly reduced the rate and extent of the rapid desensitization promoted by sustained treatment with the agonist isoproterenol. The direct contribution of Ser(345,346) in desensitization was then studied by mutating all other putative PKA and beta ARK phosphorylation sites (Ala(261,262)beta ARK(-)beta(2)AR). We found this mutant receptor to be phosphorylated upon receptor activation but not following direct activation of PKA, suggesting a role in receptor-specific (homologous) but not heterologous phosphorylation. However, despite its phosphorylated state, Ala(261,262)beta ARK(-)beta(2)AR did not undergo rapid desensitization upon agonist treatment, indicating that phosphorylation of Ser(345,346) alone is not sufficient to promote desensitization. Taken with the observation that mutation of either Ser(345,346) or of the beta ARK phosphorylation sites prevented both the hyper-phosphorylation and constitutive desensitization of a palmitoylation-less mutant (Gly(341)beta(2)AR), our data suggest a concerted/synergistic action of the two kinases that depends on the palmitoylation state of the receptor. Consistent with this notion, in vitro phosphorylation of Gly(341)beta(2)AR by the catalytic subunit of PKA facilitated further phosphorylation of the receptor by purified beta ARK. Our study therefore allows us to propose a coordinated mechanism by which sequential depalmitoylation, and phosphorylation by PKA and beta ARK lead to the functional uncoupling and desensitization of the ss(2)AR.     

Agonist-modulated palmitoylation of beta 2-adrenergic receptor in Sf9 cells.

The palmitoylation of the human beta 2-adrenergic receptor (beta 2-AR) was studied in recombinant baculovirus-infected insect Sf9 cells. At 48 h post-infection, a high level expression of an epitope-tagged beta 2-AR (10-25 pmol/mg protein) was detected by [125I]iodocyanopindolol ([125I]CYP) binding assays. The identity of the receptor was confirmed both by photoaffinity labeling and immunoblotting. The fusion receptor displayed typical beta 2-AR pharmacological properties and conferred a beta-adrenergic sensitive adenylyl cyclase activity to the Sf9 cells. Moreover, exposure of the Sf9 cells to the beta-adrenergic agonist isoproterenol induced a rapid desensitization of the receptor-stimulated adenylyl cyclase activity. Purification of the epitope-tagged beta 2-AR by immunoprecipitation as well as by alprenolol-Sepharose affinity chromatography revealed that the receptor is covalently modified with palmitic acid in the insect cells as is observed in mammalian cells. In addition, short-term incubation of the cells with isoproterenol led to a specific increase in the incorporation of [3H]palmitate in the receptor, consistent with a rapid agonist-modulated turnover of the beta 2-AR-attached palmitic acid. These results suggest that agonist-mediated regulation of beta 2-AR post-translational palmitoylation could represent an other regulatory process for G protein-coupled receptors.     

Colocalization of beta-adrenergic receptors and caveolin within the plasma membrane.

The rapid amplification of beta-adrenergic receptor signaling involves the sequential activation of multiple signaling molecules ranging from the receptor to adenylyl cyclase. The prevailing view of the agonist-induced interaction between signaling molecules is based on random collisions between proteins that diffuse freely in the plasma membrane. The recent identification of G protein alpha- and betagamma-subunits in caveolae and their functional interaction with caveolin suggests that caveolae may participate in G protein-coupled signaling. We have investigated the potential interaction of beta-adrenergic receptors with caveolin under resting conditions. beta1- and beta2-adrenergic receptors were recombinantly overexpressed in COS-7 cells. Caveolae were isolated using the detergent-free sucrose gradient centrifugation method. beta1- and beta2-adrenergic receptors were localized in the same gradient fractions as caveolin, where Gsalpha- and betagamma-subunits were detected as well. Immunofluorescence microscopy demonstrated the colocalization of beta-adrenergic receptors with caveolin, indicating a nonrandom distribution of beta-adrenergic receptors in the plasma membrane. Using polyhistidine-tagged recombinant proteins, beta-adrenergic receptors were copurified with caveolin, suggesting that they were physically bound. Our results suggest that, in addition to clathrin-coated pits, caveolae may act as another plasma membrane microdomain to compartmentalize beta-adrenergic receptors.     

Desensitization of the human beta 1-adrenergic receptor. Involvement of the cyclic AMP-dependent but not a receptor-specific protein kinase.

Human SK-N-MC neurotumor cells express beta 1- but not beta 2-adrenergic receptors. Following exposure of the cells to isoproterenol, there was no reduction in the maximum response of adenylyl cyclase to the agonist but a 3-fold shift to less sensitivity in the concentration response. This desensitization was very rapid and dose dependent; half-maximal effects occurred at 10 nM isoproterenol. A similar shift was observed when membranes from control cells were incubated with ATP and the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). No shift, however, was observed in intact cells exposed to either dibutyryl cyclic AMP or dopamine, which stimulates adenylyl cyclase in these cells through D1 dopamine receptors. To pursue the role of protein kinases in the desensitization process, cells were made permeable, loaded with a PKA inhibitor or with heparin, an inhibitor of the beta-adrenergic receptor kinase (beta ARK), and exposed to isoproterenol. The PKA inhibitor but not heparin blocked the agonist-mediated desensitization. In contrast, desensitized human tumor cells (HeLa and A431), which express beta 2-adrenergic receptors, exhibited both a shift in concentration response and a reduction in maximum response; the former was blocked by the PKA inhibitor and the latter by heparin. Our results indicated that whereas both human beta 1- and beta 2-adrenergic receptors are susceptible to PKA, only the beta 2 receptors are susceptible to beta ARK. These differences in desensitization may be due to differences in receptor structure as the human beta 1 receptor has fewer potential phosphorylation sites for beta ARK in the carboxyl terminus than the human beta 2 receptor.     

High expression of beta-adrenergic receptor kinase in human peripheral blood leukocytes. Isoproterenol and platelet activating factor can induce kinase translocation.

Receptor phosphorylation is a key step in the process of desensitization of the beta-adrenergic and other related receptors. A selective kinase (called beta-adrenergic receptor kinase, beta ARK) has been identified which phosphorylates the agonist-occupied form of the receptor. Recently the bovine beta ARK cDNA has been cloned and the highest levels of specific mRNA were found in highly innervated tissues. It was proposed that beta ARK may be primarily active on synaptic receptors. In the present study, the cDNA of human beta ARK was cloned and sequenced. The sequence was very similar to that of the bovine beta ARK (the overall amino acid homology was 98%). Very high levels of beta ARK mRNA and kinase activity were found in peripheral blood leukocytes and in several myeloid and lymphoid leukemia cell lines. Since agonist-induced beta ARK translocation is considered the first step involved in beta ARK-mediated homologous desensitization, we screened a number of G-protein-coupled receptor agonists for their ability to induce beta ARK translocation. In human mononuclear leukocytes, beta-AR agonist isoproterenol and platelet-activating factor were able to induce translocation of beta ARK from cytosol to membrane. After 20 min of exposure to isoproterenol (10 microM), the cytosolic beta ARK activity decreased to 61% of control, while membrane-associated beta ARK activity increased to 170%. 20-min exposure to platelet-activating factor (1 microM) reduced the cytosolic beta ARK activity to 42% of control with concomitant increase in membrane beta ARK activity to 214% of control. The high levels of beta ARK expression in human peripheral blood leukocytes together with the ability of isoproterenol and platelet-activating factor to induce beta ARK translocation, suggest a role for beta ARK in modulating some receptor-mediated immune functions.     

Cloning, expression, and chromosomal localization of beta-adrenergic receptor kinase 2. A new member of the receptor kinase family

The beta-adrenergic receptor kinase (beta ARK) specifically phosphorylates the agonist-occupied form of the beta-adrenergic and related G protein-coupled receptors. Structural features of this enzyme have been elucidated recently by the isolation of a cDNA that encodes bovine beta ARK. Utilizing a catalytic domain fragment of the beta ARK cDNA to screen a bovine brain cDNA library we have isolated a clone encoding a beta ARK-related enzyme which we have termed beta ARK2. Overall, this enzyme has 85% amino acid identity with beta ARK, with the protein kinase catalytic domain having 95% identity. The ability of beta ARK2 to phosphorylate various substrates was studied after expression in COS 7 cells. Although beta ARK2 is essentially equiactive with beta ARK in phosphorylating an acid-rich synthetic model peptide it was only approximately 50% as active when the substrate was the agonist-occupied beta 2-adrenergic receptor and only approximately 20% as active toward light-bleached rhodopsin. As with beta ARK, phosphorylation of the receptor substrates by beta ARK2 was completely stimulus dependent. RNA blot analysis with selected bovine tissues reveals an mRNA of 8 kilobases with a distribution similar to that of beta ARK. More detailed RNA analysis using a ribonuclease protection assay in various rat tissues suggests that the beta ARK2 message is present at much lower levels (typically 10-20%) than the beta ARK message. In the rat the beta ARK2 mRNA is localized predominantly in neuronal tissues although low levels are also observed in various peripheral tissues. The beta ARK2 gene has been localized to a region of mouse chromosome 5 whereas the beta ARK gene is localized on mouse chromosome 19. These data suggest the existence of a "family" of receptor kinases which may serve broadly to regulate receptor function.     

Phosphorylation sites on two domains of the beta 2-adrenergic receptor are involved in distinct pathways of receptor desensitization

Continuous exposure of cells to neurotransmitter or hormone agonists often results in a rapid desensitization of the cellular response. For example, pretreatment of Chinese hamster fibroblasts (CHW cells) expressing beta 2-adrenergic receptors (beta 2AR) with low (nanomolar) concentrations of isoproterenol, a beta-adrenergic agonist, causes decreases in the sensitivity of the cellular adenylyl cyclase response to the agonist, without changing the maximal responsiveness. In contrast, exposure of CHW cells to high (micromolar) concentrations of isoproterenol results in decreases in both sensitivity and the maximal responsiveness to agonist. To explore the role(s) of receptor phosphorylation in these processes, we expressed in CHW cells three mutant beta 2AR genes encoding receptors lacking putative phosphorylation sites for the cAMP-dependent protein kinase A and/or the cAMP-independent beta 2AR kinase. Using these mutants we found that exposure of cells to low concentrations of agonist appears to preferentially induce phosphorylation at protein kinase A sites. This phosphorylation correlates with the decreased sensitivity to agonist stimulation of the adenylyl cyclase response. At higher agonist concentrations phosphorylation on both the beta 2AR kinase and protein kinase A sites occurs, and only then is the maximal cyclase responsiveness elicited by agonist reduced. We conclude that low or high concentrations of agonist elicit phosphorylation of beta 2AR on distinct domains, with different implications for the functional coupling of the receptors with effector molecules.