Internalization Dissociates β2-Adrenergic Receptors

Lan, Tien Hung; Kuravi, Sudhakiranmayi; Lambert, Nevin A.

doi:10.1371/journal.pone.0017361

Cited by 54 publications

(52 citation statements)

References 35 publications

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“…4A). As was the case with G␣ s -Rluc8, stimulation with isoproterenol for 30 min decreased BRET from ␤ 2 AR-Rluc8 at the plasma membrane (25). Isoproterenol also increased BRET from ␤ 2 AR-Rluc8 at endosomes, but these increases were much larger (ϳ50 -100%) , and in cells treated with cholera toxin and then isoproterenol for 10 min.…”

Section: Resultsmentioning

confidence: 73%

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Martin

Lambert

2016

Journal of Biological Chemistry

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Heterotrimeric G proteins are localized to the plasma membrane where they transduce extracellular signals to intracellular effectors. G proteins also act at intracellular locations, and can translocate between cellular compartments. For example, G␣ s can leave the plasma membrane and move to the cell interior after activation. However, the mechanism of G␣ s translocation and its intracellular destination are not known. Here we use bioluminescence resonance energy transfer (BRET) to show that after activation, G␣ s rapidly associates with the endoplasmic reticulum, mitochondria, and endosomes, consistent with indiscriminate sampling of intracellular membranes from the cytosol rather than transport via a specific vesicular pathway. The primary source of G␣ s for endosomal compartments is constitutive endocytosis rather than activity-dependent internalization. Recycling of G␣ s to the plasma membrane is complete 25 min after stimulation is discontinued. We also show that an acylation-deacylation cycle is important for the steady-state localization of G␣ s at the plasma membrane, but our results do not support a role for deacylation in activity-dependent G␣ s internalization.Heterotrimeric G proteins are best known for their role in transducing signals from the extracellular environment across the plasma membrane (1). However, it is becoming increasingly apparent that these signaling molecules have important functions in other cellular locations (2). Therefore, it is important to know how G proteins are distributed among various intracellular compartments, and how these proteins traffic between the plasma membrane, the cytosol, cytoskeletal elements, and intracellular organelles both in their resting state and when they are activated.The most well studied example of activity-dependent G protein trafficking occurs in the retina, where intense illumination promotes the dissociation of transducin from rod outer segment disks and the subsequent translocation of both G␣ t and G␤␥ into the rod inner segment (3). Activity-dependent translocation of G␣ and G␤␥ subunits occurs in other cells as well (4, 5), most notably for G␣ s , the subunit responsible for stimulation of adenylate cyclase. Stimulation of a cognate receptor or inhibition of GTPase activity promotes activation and translocation of G␣ s from the plasma membrane to the cell interior (6 -8).One important yet poorly understood aspect of G␣ s internalization is the intracellular destination of this G protein. Cell fractionation studies have shown that a large amount of constitutively active G␣ s is found in the soluble fraction, suggesting that active G␣ s may simply become soluble in the cytosol (8, 9). However, only a small amount of G␣ s enters the soluble fraction after receptor-mediated activation, consistent with vesicle-mediated internalization and continued association with membranes. Imaging studies have shown that internalized G␣ s can appear to be diffuse in the cytosol (8, 9), but association with intracellular vesicles has also been observed (10 -12)...

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Section: Resultsmentioning

confidence: 73%

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Martin

Lambert

2016

Journal of Biological Chemistry

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“…The significantly reduced proportion of receptor dimers indicates, however, that remodeling of the β‐adrenergic signaling has already occurred during this short period independent of the 2 intervention strategies. A reduction in the receptor dimers in turn leads to a lessening of the signal transduction and to accelerated receptor internalization 33, 34, 35, 36…”

Section: Discussionmentioning

confidence: 99%

Adverse Effects on β‐Adrenergic Receptor Coupling: Ischemic Postconditioning Failed to Preserve Long‐Term Cardiac Function

Schreckenberg

Bencsik

Weber

et al. 2017

JAHA

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BackgroundIschemic preconditioning (IPC) and ischemic postconditioning (IPoC) are currently among the most efficient strategies protecting the heart against ischemia/reperfusion injury. However, the effect of IPC and IPoC on functional recovery following ischemia/reperfusion is less clear, particularly with regard to the specific receptor‐mediated signaling of the postischemic heart. The current article examines the effect of IPC or IPoC on the regulation and coupling of β‐adrenergic receptors and their effects on postischemic left ventricular function.Methods and ResultsThe β‐adrenergic signal transduction was analyzed in 3‐month‐old Wistar rats for each of the intervention strategies (Sham, ischemia/reperfusion, IPC, IPoC) immediately and 7 days after myocardial infarction. Directly after the infarction a cardioprotective potential was demonstrated for both IPC and IPoC: the infarct size was reduced, apoptosis and production of reactive oxygen species were lowered, and the myocardial tissue was preserved. Seven days after myocardial ischemia, only IPC hearts showed significant functional improvement. Along with a deterioration in fractional shortening, IPoC hearts no longer responded adequately to β‐adrenergic stimulation. The stabilization of β‐adrenergic receptor kinase‐2 via increased phosphorylation of Mdm2 (an E3‐ubiquitin ligase) was responsible for desensitization of β‐adrenergic receptors and identified as a characteristic specific to IPoC hearts.ConclusionsImmediately after myocardial infarction, rapid and transient activation of β‐adrenergic receptor kinase‐2 may be an appropriate means to protect the injured heart from excessive stress. In the long term, however, induction and stabilization of β‐adrenergic receptor kinase‐2, with the resultant loss of positive inotropic function, leads to the functional picture of heart failure.

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“…4). The GFP-KRAS fusion protein of the last 25 amino acid (aa) (RKHKEKMSKDGKKKKKKSKTKCVIM, including the farnesylation site) of KRAS and the fluorescent protein GFP was provided by Nevin A. Lambert (Lan et al, 2011). FLAG-epitope-tagged DVL1 was from Madelon M. Maurice (University Medical Center, Utrecht, The Netherlands), DVL2-MYC was from S. A. Yanagawa (Kyoto University, Kyoto, Japan), and DVL3-FLAG was from Randall T. Moon (University of Washington School of Medicine, Seattle, WA).…”

Section: Methodsmentioning

confidence: 99%

WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα_12/13

et al. 2016

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Frizzleds (FZDs) are unconventional G protein-coupled receptors that belong to the class Frizzled. They are bound and activated by the Wingless/Int-1 lipoglycoprotein (WNT) family of secreted lipoglycoproteins. To date, mechanisms of signal initiation and FZD-G protein coupling remain poorly understood. Previously, we showed that FZD 6 assembles with Ga i1 /Ga q (but not with Ga s , Ga o and Ga 12/13 ), and that these inactive-state complexes are dissociated by WNTs and regulated by the phosphoprotein Dishevelled (DVL). Here, we investigated the inactive-state assembly of heterotrimeric G proteins with FZD 4 , a receptor important in retinal vascular development and frequently mutated in Norrie disease or familial exudative vitreoretinopathy. Live-cell imaging experiments using fluorescence recovery after photobleaching show that human FZD 4 assembles-in a DVL-independent manner-with Ga 12/13 but not representatives of other heterotrimeric G protein subfamilies, such as Ga i1 , Ga o , Ga s , and Ga q . The FZD 4 -G protein complex dissociates upon stimulation with WNT-3A, WNT-5A, WNT-7A, and WNT-10B. In addition, WNT-induced dynamic mass redistribution changes in untransfected and, even more so, in FZD 4 green fluorescent protein-transfected cells depend on Ga 12/13 . Furthermore, expression of FZD 4 and Ga 12 or Ga 13 in human embryonic kidney 293 cells induces WNT-dependent membrane recruitment of p115-RHOGEF (RHO guanine nucleotide exchange factor, molecular weight 115 kDa), a direct target of Ga 12/13 signaling, underlining the functionality of an FZD 4 -Ga 12/13 -RHO signaling axis. In summary, Ga 12/13 -mediated WNT/FZD 4 signaling through p115-RHOGEF offers an intriguing and previously unappreciated mechanistic link of FZD 4 signaling to cytoskeletal rearrangements and RHO signaling with implications for the regulation of angiogenesis during embryonic and tumor development.

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Internalization Dissociates β2-Adrenergic Receptors

Cited by 54 publications

References 35 publications

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Adverse Effects on β‐Adrenergic Receptor Coupling: Ischemic Postconditioning Failed to Preserve Long‐Term Cardiac Function

WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα_12/13

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Internalization Dissociates β2-Adrenergic Receptors

Cited by 54 publications

References 35 publications

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Activated G Protein Gαs Samples Multiple Endomembrane Compartments

Adverse Effects on β‐Adrenergic Receptor Coupling: Ischemic Postconditioning Failed to Preserve Long‐Term Cardiac Function

WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα12/13

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WNT Stimulation Dissociates a Frizzled 4 Inactive-State Complex with Gα_12/13