Beta AR Internalization Research Articles

Abstract 13074: Impact of Adrenergic Receptor Polymorphisms on Incident Ventricular Fibrillation During Acute Myocardial Ischemia: The MAP-MI Study

Rationale: During acute coronary syndromes (ACS) adrenergic receptor (AR) signaling may have major effects on cardiac arrhythmias including VF. AR gene products are highly polymorphic, and minor variants exhibit potentially important pharmacologic differences compared to receptors encoded by major allele homozygotes ( mah ). Objective: We tested the general hypothesis that polymorphic variants in the ADRB1 , ADRB2 or ADRA2C genes affect incident VF during ACS. Methods and Results: We recruited 953 subjects with a proven MI and no previous history of AC. In a case-control design 477 VF patients were compared to 476 controls without VF who did not differ in baseline characteristics. DNA collection and subsequent genotyping for ADRB1 389/49, ADRB2 27/16 and ADRA2C 322-325 Ins/Del were available in 718-779 patients. Results were expressed as Odds Ratios (OR) for VF vs. No-VF subjects by comparing each mah to minor allele monotype genotypes, genotype combinations or haplotypes. The entire cohort OR (95% CI) for patients receiving (41%) vs. not receiving beta-blockade (BB) was (0.70 (0.53, 0.93), P=0.014), dictating a BB adjustment in the genotype-VF analyses. No minor allele genotype was associated with increased incident VF. Monotypes with statistically significant ORs and their mah comparators were: ADRB2 monotypes and ADRB1 or ADRB2 haplotypes were not associated with VF reduction. Two of 3 ADRB1 and 3 of 4 ADRB2 monotypes or genotype combinations associated with VF reduction encoded ARs that exhibit increased agonist-induced internalization. Conclusions: In acute MI AR variants associated with reduced incident VF are those that mediate increased adrenergic drive ( ADRA2 C Del) and beta-AR internalization. This combination would be predicted to redirect beta-AR signaling from canonical pathways to "biased" EGFR and ERK1/2 mediated cardioprotection.

Quantitative Modeling of GRK-Mediated β2AR Regulation

We developed a unified model of the GRK-mediated β2 adrenergic receptor (β2AR) regulation that simultaneously accounts for six different biochemical measurements of the system obtained over a wide range of agonist concentrations. Using a single deterministic model we accounted for (1) GRK phosphorylation in response to various full and partial agonists; (2) dephosphorylation of the GRK site on the β2AR; (3) β2AR internalization; (4) recycling of the β2AR post isoproterenol treatment; (5) β2AR desensitization; and (6) β2AR resensitization. Simulations of our model show that plasma membrane dephosphorylation and recycling of the phosphorylated receptor are necessary to adequately account for the measured dephosphorylation kinetics. We further used the model to predict the consequences of (1) modifying rates such as GRK phosphorylation of the receptor, arrestin binding and dissociation from the receptor, and receptor dephosphorylation that should reflect effects of knockdowns and overexpressions of these components; and (2) varying concentration and frequency of agonist stimulation “seen” by the β2AR to better mimic hormonal, neurophysiological and pharmacological stimulations of the β2AR. Exploring the consequences of rapid pulsatile agonist stimulation, we found that although resensitization was rapid, the β2AR system retained the memory of the previous stimuli and desensitized faster and much more strongly in response to subsequent stimuli. The latent memory that we predict is due to slower membrane dephosphorylation, which allows for progressive accumulation of phosphorylated receptor on the surface. This primes the receptor for faster arrestin binding on subsequent agonist activation leading to a greater extent of desensitization. In summary, the model is unique in accounting for the behavior of the β2AR system across multiple types of biochemical measurements using a single set of experimentally constrained parameters. It also provides insight into how the signaling machinery can retain memory of prior stimulation long after near complete resensitization has been achieved.

β-Arrestin-biased Agonism at the β2-Adrenergic Receptor

Classically, the beta 2-adrenergic receptor (beta 2AR) and other members of the seven-transmembrane receptor (7TMR) superfamily activate G protein-dependent signaling pathways in response to ligand stimulus. It has recently been discovered, however, that a number of 7TMRs, including beta 2AR, can signal via beta-arrestin-dependent pathways independent of G protein activation. It is currently unclear if among beta 2AR agonists there exist ligands that disproportionately signal via G proteins or beta-arrestins and are hence "biased." Using a variety of approaches that include highly sensitive fluorescence resonance energy transfer-based methodologies, including a novel assay for receptor internalization, we show that the majority of known beta 2AR agonists exhibit relative efficacies for beta-arrestin-associated activities (beta-arrestin membrane translocation and beta 2AR internalization) identical to the irrelative efficacies for G protein-dependent signaling (cyclic AMP generation). However, for three betaAR ligands there is a marked bias toward beta-arrestin signaling; these ligands stimulate beta-arrestin-dependent receptor activities to a much greater extent than would be expected given their efficacy for G protein-dependent activity. Structural comparison of these biased ligands reveals that all three are catecholamines containing an ethyl substitution on the alpha-carbon, a motif absent on all of the other, unbiased ligands tested. Thus, these studies demonstrate the potential for developing a novel class of 7TMR ligands with a distinct bias for beta-arrestin-mediated signaling.

Protein kinase activity of phosphoinositide 3-kinase regulates β-adrenergic receptor endocytosis

Phosphoinositide 3-kinase (PI(3)K) is a unique enzyme characterized by both lipid and protein kinase activities. Here, we demonstrate a requirement for the protein kinase activity of PI(3)K in agonist-dependent beta-adrenergic receptor (betaAR) internalization. Using PI(3)K mutants with either protein or lipid phosphorylation activity, we identify the cytoskeletal protein non-muscle tropomyosin as a substrate of PI(3)K, which is phosphorylated in a wortmannin-sensitive manner on residue Ser 61. A constitutively dephosphorylated (S61A) tropomyosin mutant blocks agonist-dependent betaAR internalization, whereas a tropomyosin mutant that mimics constitutive phosphorylation (S61D) complements the PI(3)K mutant, with only lipid phosphorylation activity reversing the defective betaAR internalization. Notably, knocking down endogenous tropomyosin expression using siRNAs that target different regions if tropomyosin resulted in complete inhibition of betaAR endocytosis, showing that non-muscle tropomyosin is essential for agonist-mediated receptor internalization. These studies demonstrate a previously unknown role for the protein phosphorylation activity of PI(3)K in betaAR internalization and identify non-muscle tropomyosin as a cellular substrate for protein kinase activity of PI(3)K.

Inhibition of receptor-localized PI3K preserves cardiac beta-adrenergic receptor function and ameliorates pressure overload heart failure.

beta-Adrenergic receptor (betaAR) downregulation and desensitization are hallmarks of the failing heart. However, whether abnormalities in betaAR function are mechanistically linked to the cause of heart failure is not known. We hypothesized that downregulation of cardiac betaARs can be prevented through inhibition of PI3K activity within the receptor complex, because PI3K is necessary for betaAR internalization. Here we show that in genetically modified mice, disrupting the recruitment of PI3K to agonist-activated betaARs in vivo prevents receptor downregulation in response to chronic catecholamine administration and ameliorates the development of heart failure with pressure overload. Disruption of PI3K/betaAR colocalization is required to preserve betaAR signaling, since deletion of a single PI3K isoform (PI3Kgamma knockout) is insufficient to prevent the recruitment of other PI3K isoforms and subsequent betaAR downregulation with catecholamine stress. These data demonstrate a specific role for receptor-localized PI3K in the regulation of betaAR turnover and show that abnormalities in betaAR function are associated with the development of heart failure. Thus, a strategy that blocks the membrane translocation of PI3K and leads to the inhibition of betaAR-localized PI3K activity represents a novel therapeutic approach to restore normal betaAR signaling and preserve cardiac function in the pressure overloaded failing heart.

Characterization of G protein-coupled receptor regulation in antisense mRNA-expressing cells with reduced arrestin levels.

Previous studies with overexpressing wild-type or dominant negative nonvisual arrestins have established a role for these proteins in beta2-adrenergic receptor (beta2AR) internalization, desensitization, and resensitization. To validate and extend such findings, we employed an antisense strategy to target the nonvisual arrestins, arrestin-2 and arrestin-3, and determined the associated effects on the regulation of G protein-coupled receptor (GPCR) signaling. HEK293 cells stably expressing antisense constructs targeting arrestin-2 exhibited a selective reduction (approximately 50%) in arrestin-2 levels, while arrestin-3 antisense constructs resulted in reductions (>/=50%) in both arrestin-2 and arrestin-3 levels. Initial analysis of these cells demonstrated that a reduced level of arrestin expression resulted in a significant decrease in the extent of agonist-induced internalization of exogenously expressed beta2ARs, but had no effect on internalization of either m2 or m3 muscarinic acetylcholine receptors. Additional characterization involved assessing the role of arrestins in the regulation of endogenous GPCRs in these cells. Reduced arrestin levels significantly decreased the rate of endogenous beta2AR internalization, desensitization, and resensitization. Further analysis demonstrated that the desensitization of endogenous A2b adenosine and prostaglandin E2-stimulated receptors was also attenuated in cells with reduced arrestin levels. The effects on the beta2-adrenergic, A2b adenosine, and PGE2-stimulated receptors were similar among cell lines that exhibited either a selective reduction in arrestin-2 levels or a reduction in both arrestin-2 and -3 levels. These findings establish the utility of antisense approaches in the examination of arrestin-mediated GPCR regulation.

Identification of NSF as a β-Arrestin1-binding Protein: IMPLICATIONS FOR β2-ADRENERGIC RECEPTOR REGULATION

Previous studies have demonstrated that beta-arrestin1 serves to target G protein-coupled receptors for internalization via clathrin-coated pits and that its endocytic function is regulated by dephosphorylation at the plasma membrane. Using the yeast two-hybrid system, we have identified a novel beta-arrestin1-binding protein, NSF (N-ethylmaleimide-sensitive fusion protein), an ATPase essential for many intracellular transport reactions. We demonstrate that purified recombinant beta-arrestin1 and NSF interact in vitro and that these proteins can be coimmunoprecipitated from cells. beta-Arrestin1-NSF complex formation exhibits a conformational dependence with beta-arrestin1 preferentially interacting with the ATP bound form of NSF. In contrast to the beta-arrestin1-clathrin interaction, however, the phosphorylation state of beta-arrestin1 does not affect NSF binding. Functionally, overexpression of NSF in HEK 293 cells significantly enhances agonist-mediated beta2-adrenergic receptor (beta2-AR) internalization. Furthermore, when coexpressed with a beta-arrestin1 mutant (betaarr1S412D) that mimics a constitutively phosphorylated form of beta-arrestin1 and that acts as a dominant negative with regards to beta2-AR internalization, NSF rescues the betaarr1S412D-mediated inhibition of beta2-AR internalization. The demonstration of beta-arrestin1-NSF complex formation and the functional consequences of NSF overexpression suggest a hitherto unappreciated role for NSF in facilitating clathrin coat-mediated G protein-coupled receptor internalization.

Src-mediated Tyrosine Phosphorylation of Dynamin Is Required for β2-Adrenergic Receptor Internalization and Mitogen-activated Protein Kinase Signaling

Some forms of G protein-coupled receptor signaling, such as activation of mitogen-activated protein kinase cascade as well as resensitization of receptors after hormone-induced desensitization, require receptor internalization via dynamin-dependent clathrin-coated pit mechanisms. Here we demonstrate that activation of beta2-adrenergic receptors (beta2-ARs) leads to c-Src-mediated tyrosine phosphorylation of dynamin, which is required for receptor internalization. Two tyrosine residues, Tyr231 and Tyr597, are identified as the major phosphorylation sites. Mutation of these residues to phenylalanine dramatically decreases the c-Src-mediated phosphorylation of dynamin following beta2-AR stimulation. Moreover, expression of Y231F/Y597F dynamin inhibits beta2-AR internalization and the isoproterenol-stimulated mitogen-activated protein kinase activation. Thus, agonist-induced, c-Src-mediated tyrosine phosphorylation of dynamin is essential for its function in clathrin mediated G protein-coupled receptor endocytosis.

Role of Clathrin-mediated Endocytosis in Agonist-induced Down-regulation of the β2-Adrenergic Receptor

Previous studies have demonstrated that non-visual arrestins function as adaptors in clathrin-mediated endocytosis to promote agonist-induced internalization of the beta2-adrenergic receptor (beta2AR). Here, we characterized the effects of arrestins and other modulators of clathrin-mediated endocytosis on down-regulation of the beta2AR. In COS-1 and HeLa cells, non-visual arrestins promote agonist-induced internalization and down-regulation of the beta2AR, whereas dynamin-K44A, a dominant-negative mutant of dynamin that inhibits clathrin-mediated endocytosis, attenuates beta2AR internalization and down-regulation. In HEK293 cells, dynamin-K44A profoundly inhibits agonist-induced internalization and down-regulation of the beta2AR, suggesting that receptor internalization is critical for down-regulation in these cells. Moreover, a dominant-negative mutant of beta-arrestin, beta-arrestin-(319-418), also inhibits both agonist-induced receptor internalization and down-regulation. Immunofluorescence microscopy analysis reveals that the beta2AR is trafficked to lysosomes in HEK293 cells, where presumably degradation of the receptor occurs. These studies demonstrate that down-regulation of the beta2AR is in part due to trafficking of the beta2AR via the clathrin-coated pit endosomal pathway to lysosomes.

Modulation of the Arrestin-Clathrin Interaction in Cells: CHARACTERIZATION OF β-ARRESTIN DOMINANT-NEGATIVE MUTANTS

We recently demonstrated that nonvisual arrestins interact via a C-terminal binding domain with clathrin and function as adaptor proteins to promote beta2-adrenergic receptor (beta2AR) internalization. Here, we investigated the potential utility of a mini-gene expressing the clathrin-binding domain of beta-arrestin (beta-arrestin (319-418)) to function as a dominant-negative with respect to beta2AR internalization and compared its properties with those of beta-arrestin and beta-arrestin-V53D, a previously reported dominant-negative mutant. In vitro studies demonstrated that beta-arrestin-V53D bound better to clathrin than beta-arrestin but was significantly impaired in its interaction with phosphorylated G protein-coupled receptors. In contrast, whereas beta-arrestin (319-418) also bound well to clathrin it completely lacked receptor binding activity. When coexpressed with the beta2AR in HEK293 cells, beta-arrestin (319-418) effectively inhibited agonist-promoted receptor internalization, whereas beta-arrestin-V53D was only modestly effective. However, both constructs significantly inhibited the stimulation of beta2AR internalization by beta-arrestin in COS-1 cells. Interestingly, immunofluorescence microscopy analysis reveals that both beta-arrestin (319-418) and beta-arrestin-V53D are constitutively localized in clathrin-coated pits in COS-1 cells. These results indicate the potential usefulness of beta-arrestin (319-418) to effectively block arrestin-clathrin interaction in cells and suggest that this construct may prove useful in further defining the mechanisms involved in G protein-coupled receptor trafficking.

Beta-arrestin acts as a clathrin adaptor in endocytosis of the beta2-adrenergic receptor.

The ability of a system to regulate its responsiveness in the presence of a continuous stimulus, often termed desensitization, has been extensively characterized for the beta2-adrenergic receptor (beta2AR). beta2AR signalling is rapidly attenuated through receptor phosphorylation and subsequent binding of the protein beta-arrestin. Ultimately the receptor undergoes internalization, and although the molecular mechanism is unclear, receptor phosphorylation and beta-arrestin binding have been implicated in this processs. Here we report that beta-arrestin and arrestin-3, but not visual arrestin, promote beta2AR internalization and bind with high affinity directly and stoichiometrically to clathrin, the major structural protein of coated pits. Moreover, beta-arrestin/arrestin chimaeras that are defective in either beta2AR or clathrin binding show a reduced ability to promote beta2AR endocytosis. Immunofluorescence microscopy of intact cells indicates an agonist-dependent colocalization of the beta2AR and beta-arrestin with clathrin. These results show that beta-arrestin functions as an adaptor in the receptor-mediated endocytosis pathway, and suggest a general mechanism for regulating the trafficking of G-protein-coupled receptors.