Subtype-Specific Intracellular Trafficking of α<sub>2</sub>-Adrenergic Receptors

Daunt, David A.; Hurt, Carl M.; Hein, Lutz; Kallio, Johanna; Feng, Felix Y.; Kobilka, Brian K.

doi:10.1124/mol.51.5.711

Cited by 183 publications

(191 citation statements)

References 22 publications

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“…Prolonged incubation of the cells with UK14304 did not influence ␣ 2 -AR expression or [ 125 I]VIP binding at the cell surface. Incubation of cells with the ␣ 2 -AR agonist (18 h) also did not alter the ability of the ␣ 2 -AR to mediate inhibition of VIP-induced increases in cAMP, 5 reflecting the limited ability of this ␣ 2 -AR subtype to undergo desensitization and down-regulation (36,37).…”

Section: Resultsmentioning

confidence: 99%

AGS3 and Signal Integration by Gαs- and Gαi-coupled Receptors

Sato

Gettys

Lanier

2004

Journal of Biological Chemistry

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AGS3-LONG and AGS3-SHORT contain G-protein regulatory motifs that interact with and stabilize the GDPbound conformation of G␣ i > G␣ o . AGS3 and related proteins may influence signal strength or duration as well as the adaptation of the signaling system associated with sustained stimulation. To address these issues, we determined the effect of AGS3 on the integration of stimulatory (G␣ s -mediated vasoactive intestinal peptide receptor) and inhibitory (G␣ i -mediated ␣ 2 -adrenergic receptor (␣ 2 -AR)) signals to adenylyl cyclase in Chinese hamster ovary cells. AGS3-SHORT and AGS3-LONG did not alter the VIP-induced increase in cAMP or the inhibitory effect of ␣ 2 -AR activation. System adaptation was addressed by determining the influence of AGS3 on the sensitization of adenylyl cyclase that occurs following prolonged activation of a G␣ i -coupled receptor. Incubation of cells with the ␣ 2 -AR agonist UK14304 (1 M) for 18 h resulted in a ϳ1.8-fold increase in the vasoactive intestinal peptide-induced activation of adenylyl cyclase, and this was associated with a decrease in membrane-associated G␣ i3 . Both effects were blocked by AGS3-SHORT. AGS3-SHORT also decreased the rate of G␣ i3 decay. A mutant AGS3-SHORT incapable of binding G-protein was inactive. These data suggest that AGS3 and perhaps other G-protein regulatory motif-containing proteins increase the stability of G␣ i in the membrane, which influences the adaptation of the cell to prolonged activation of G␣ i -coupled receptors.The activation/deactivation cycle for heterotrimeric G-protein involves the exchange of GDP for GTP and the subsequent hydrolysis of GTP by G␣ subunits. These events are generally initiated by a G-protein-coupled receptor at the cell surface. However, increasing evidence indicates the existence of unexpected regulatory mechanisms for the G-protein activation/ deactivation cycle and a role for G-protein subunits in signaling events within the cell that do not involve a cell-surface receptor (1). These unexpected regulatory mechanisms include accessory proteins that accelerate GTPase activity, serve as novel binding partners for G␣ and G␤␥ subunits, and/or regulate guanine nucleotide exchange independent of a G-protein coupled receptor (1-6).

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Section: Resultsmentioning

confidence: 99%

AGS3 and Signal Integration by Gαs- and Gαi-coupled Receptors

Sato

Gettys

Lanier

2004

Journal of Biological Chemistry

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“…Receptor internalization was measured by cell surface ELISA as previously described (46). Detection of ERK Phosphorylation and EGFR Transactivation.…”

Section: Methodsmentioning

confidence: 99%

β-arrestin–biased signaling through the β ₂ -adrenergic receptor promotes cardiomyocyte contraction

Carr

Schilling

Song

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

103

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β-adrenergic receptors (βARs) are critical regulators of acute cardiovascular physiology. In response to elevated catecholamine stimulation during development of congestive heart failure (CHF), chronic activation of G s -dependent β 1 AR and G i -dependent β 2 AR pathways leads to enhanced cardiomyocyte death, reduced β 1 AR expression, and decreased inotropic reserve. β-blockers act to block excessive catecholamine stimulation of βARs to decrease cellular apoptotic signaling and normalize β 1 AR expression and inotropy. Whereas these actions reduce cardiac remodeling and mortality outcomes, the effects are not sustained. Converse to G-protein-dependent signaling, β-arrestin-dependent signaling promotes cardiomyocyte survival. Given that β 2 AR expression is unaltered in CHF, a β-arrestin-biased agonist that operates through the β 2 AR represents a potentially useful therapeutic approach. Carvedilol, a currently prescribed nonselective β-blocker, has been classified as a β-arrestin-biased agonist that can inhibit basal signaling from βARs and also stimulate cell survival signaling pathways. To understand the relative contribution of β-arrestin bias to the efficacy of select β-blockers, a specific β-arrestin-biased pepducin for the β 2 AR, intracellular loop (ICL)1-9, was used to decouple β-arrestin-biased signaling from occupation of the orthosteric ligand-binding pocket. With similar efficacy to carvedilol, ICL1-9 was able to promote β 2 AR phosphorylation, β-arrestin recruitment, β 2 AR internalization, and β-arrestin-biased signaling. Interestingly, ICL1-9 was also able to induce β 2 AR-and β-arrestin-dependent and Ca 2+ -independent contractility in primary adult murine cardiomyocytes, whereas carvedilol had no efficacy. Thus, ICL1-9 is an effective tool to access a pharmacological profile stimulating cardioprotective signaling and inotropic effects through the β 2 AR and serves as a model for the next generation of cardiovascular drug development.B eta-antagonists, also known as β-blockers, have been indicated for the treatment of pathological cardiac diseases, including congestive heart failure (CHF) and high blood pressure, for decades (1, 2). A select number of these agents, including the clinically used carvedilol, have been identified as β-arrestinbiased agonists of β-adrenergic receptors based on their ability to promote β-arrestin-dependent signaling over G-protein activation (3, 4). It is believed that the β-arrestin activation may provide additional cardioprotection based on its ability to mediate antiapoptotic signaling. As these are orthosteric ligands, there have been no means to decouple the activation of receptor-dependent β-arrestin signaling from the occupation of the orthosteric ligandbinding pocket to study their independent contribution to its efficacy as these properties appear inherently linked.Recently, we described the characterization of a library of modulators of the β 2 -adrenergic receptor (β 2 AR) known as pepducins (5). Pepducins are lipidated peptides derived from the intracel...

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“…Cells were washed twice with TBS and blocked for 45 min with blocking buffer (TBS, 1% BSA, and 1 mM CaCl 2 ) at room temperature. Cell surface FLAG-␤ 2 AR was detected by ELISA as described previously (30). Briefly, cells were incubated with rabbit polyclonal anti-FLAG (Sigma) for 1 h at room temperature, anti-rabbit HRP secondary antibody (Vector Laboratories) for 1 h at room temperature, and then washed twice with cold blocking buffer, developed by adding a one-step 2,2Ј-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate (Thermo Scientific), and incubated at room temperature for 25 min.…”

Section: Methodsmentioning

confidence: 99%

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists*

Carr

Quoyer

et al. 2014

Journal of Biological Chemistry

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Background: A G s -biased agonist for the ␤ 2 -adrenergic receptor (␤ 2 AR) has yet to be reported. Results: A screen of ␤ 2 AR pepducins identified receptor-dependent and receptor-independent pepducins that selectively activate G s . Conclusion: Receptor-dependent pepducins promote a G s -biased conformation of the ␤ 2 AR, whereas receptor-independent pepducins directly activate G s . Significance: G s -biased pepducins provide a valuable tool for the continued study of ␤ 2 AR function and may prove useful as next-generation asthma therapeutics.

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Subtype-Specific Intracellular Trafficking of α₂-Adrenergic Receptors

Cited by 183 publications

References 22 publications

AGS3 and Signal Integration by Gαs- and Gαi-coupled Receptors

AGS3 and Signal Integration by Gαs- and Gαi-coupled Receptors

β-arrestin–biased signaling through the β ₂ -adrenergic receptor promotes cardiomyocyte contraction

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists*

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Subtype-Specific Intracellular Trafficking of α2-Adrenergic Receptors

Cited by 183 publications

References 22 publications

AGS3 and Signal Integration by Gαs- and Gαi-coupled Receptors

AGS3 and Signal Integration by Gαs- and Gαi-coupled Receptors

β-arrestin–biased signaling through the β 2 -adrenergic receptor promotes cardiomyocyte contraction

Development and Characterization of Pepducins as Gs-biased Allosteric Agonists*

Contact Info

Product

Resources

About

Subtype-Specific Intracellular Trafficking of α₂-Adrenergic Receptors

β-arrestin–biased signaling through the β ₂ -adrenergic receptor promotes cardiomyocyte contraction