G protein coupled receptor signaling complexes in live cells

Hepler, John R.

doi:10.4161/cl.29392

Cited by 11 publications

(10 citation statements)

References 33 publications

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“…Our initial studies attempted this using a live cell BRET assay with luciferase-tagged Gai (Gai-Luc) and a Venustagged Gbc (Gbc-Ven), activated with an appropriate Gai/o-coupled GPCR. However, we were unable to detect significant heterotrimer dissociation following receptor activation using this assay (data not shown), consistent with previous reports of G protein heterotrimer rearrangement in cells (Bunemann et al 2003;Hepler 2014).…”

Section: Resultssupporting

confidence: 92%

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RGS14 regulates the lifetime of Gα‐GTP signaling but does not prolong Gβγ signaling following receptor activation in live cells

Brown

Lambert

Hepler

2016

Pharmacology Res & Perspec

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RGS14 is a multifunctional scaffolding protein possessing two distinct G protein interaction sites including a regulator of G protein signaling (RGS) domain that acts as a GTPase activating protein (GAP) to deactivate Gαi/o‐GTP proteins, and a G protein regulatory (GPR) motif that binds inactive Gαi1/3‐GDP proteins independent of Gβγ. GPR interactions with Gαi recruit RGS14 to the plasma membrane to interact with Gαi‐linked GPCRs and regulate Gαi signaling. While RGS14 actions on Gα proteins are well characterized, consequent effects on Gβγ signaling remain unknown. Conventional RGS proteins act as dedicated GAPs to deactivate Gα and Gβγ signaling following receptor activation. RGS14 may do the same or, alternatively, may coordinate its actions to deactivate Gα‐GTP with the RGS domain and then capture the same Gα‐GDP via its GPR motif to prevent heterotrimer reassociation and prolong Gβγ signaling. To test this idea, we compared the regulation of G protein activation and deactivation kinetics by a conventional RGS protein, RGS4, and RGS14 in response to GPCR agonist/antagonist treatment utilizing bioluminescence resonance energy transfer (BRET). Co‐expression of either RGS4 or RGS14 inhibited the release of free Gβγ after agonist stimulation and increased the deactivation rate of Gα, consistent with their roles as GTPase activating proteins (GAPs). Overexpression of inactive Gαi1 to recruit RGS14 to the plasma membrane did not alter RGS14′s capacity to act as a GAP for a second Gαo protein. These results demonstrate the role of RGS14 as a dedicated GAP and suggest that the G protein regulatory (GPR) motif functions independently of the RGS domain and is silent in regulating GAP activity in a cellular context.

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Section: Resultssupporting

confidence: 92%

“…However, we were unable to detect significant heterotrimer dissociation following receptor activation using this assay (data not shown), consistent with previous reports of G protein heterotrimer rearrangement in cells (Bunemann et al. ; Hepler ).…”

Section: Resultssupporting

confidence: 92%

RGS14 regulates the lifetime of Gα‐GTP signaling but does not prolong Gβγ signaling following receptor activation in live cells

Brown

Lambert

Hepler

2016

Pharmacology Res & Perspec

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“…from three independent experiments performed in duplicate. The K B values of MRS2500 and BPTU and slopes from Schild analysis from three separate experiments are listed in Table 1. residence time (Guo et al, 2014;Klein Herenbrink et al, 2016), signaling amplification (Hepler, 2014), probe dependence (Kenakin, 2008;Ehlert, 2013), and cell background (Kenakin, 2009), in addition to the possibility of the conformational selective antagonism of BPTU in different pathways (Edelstein and Changeux, 2016).…”

Section: Resultsmentioning

confidence: 99%

“…Allosteric sites, distinct conformations, and receptor internalization have also been used as alternative explanations. Insurmountable antagonism is also related to signaling amplification (Hepler, 2014), probe dependence (Ehlert, 2013), cell types. and cell background (Kenakin, 2009).…”

Section: Discussionmentioning

confidence: 99%

Distinct Signaling Patterns of Allosteric Antagonism at the P2Y₁ Receptor

Gao

2017

Mol Pharmacol

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Traditionally, G protein-coupled receptor antagonists are classified as competitive or noncompetitive and surmountable or insurmountable based on functional antagonism. P2Y 1 receptor (P2Y 1 R) structures showed two antagonists binding to two spatially distinct sites: nucleotide MRS2500 (orthosteric, contacting the helical bundle) and urea BPTU (allosteric, on the external receptor surface). However, the nature of their P2Y 1 R antagonism has not been characterized. Here we characterized BPTU antagonism at various signaling pathways activated by structurally diverse agonists. BPTU rightward shifted the concentration-response curves of both 2-methylthioadenosine 59-diphosphate trisodium salt and MRS2365 (59-diphosphates) in some signaling events, such as extracellular signal-regulated kinase 1/2 and label free, in a parallel manner without affecting the maximum agonist effect (E max ) but antagonized insurmountably (suppressed agonist E max ) in signaling events such as guanosine 59-3-O-(thio)triphosphate binding and b-arrestin2 recruitment. However, with dinucleotide Ap4A as an agonist, BPTU suppressed the E max insurmountably in all signaling pathways. By comparison, MRS2500 behaved as surmountable antagonist rightward-shifting concentration-response curves of all three agonists in a parallel manner for all signaling pathways measured. Thus, we demonstrated a previously undocumented phenomenon that P2Y 1 R antagonism patterns could vary in different signaling pathways, which could be related to conformational selection, signaling amplification, and probe dependence. This phenomenon may apply generally to other receptors considering that antagonism by a specific ligand is often not compared at multiple signaling pathways. Thus, antagonism can be surmountable or insurmountable depending on the signaling pathways measured and the agonists used, which should be of broad relevance to drug discovery and disease treatment.

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“…In general terms, the canonical mechanism of heterotrimeric G protein signaling involves redistribution of GTP-bound Ga and Gbg at the internal surface of the plasma membrane (Lambert, 2008;Hepler, 2014). There, they interact with their effectors, which are either integral plasma membrane proteins or cytosolic signaling partners recruited via direct interactions.…”

Section: Availability Of Gbg For Chemotactic Signalingmentioning

confidence: 99%

Gβγ Pathways in Cell Polarity and Migration Linked to Oncogenic GPCR Signaling: Potential Relevance in Tumor Microenvironment

et al. 2016

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Cancer cells and stroma cells in tumors secrete chemotactic agonists that exacerbate invasive behavior, promote tumor-induced angiogenesis, and recruit protumoral bone marrow-derived cells. In response to shallow gradients of chemotactic stimuli recognized by G protein-coupled receptors (GPCRs), Gβγ-dependent signaling cascades contribute to specifying the spatiotemporal assembly of cytoskeletal structures that can dynamically alter cell morphology. This sophisticated process is intrinsically linked to the activation of Rho GTPases and their cytoskeletal-remodeling effectors. Thus, Rho guanine nucleotide exchange factors, the activators of these molecular switches, and their upstream signaling partners are considered participants of tumor progression. Specifically, phosphoinositide-3 kinases (class I PI3Ks, β and γ) and P-Rex1, a Rac-specific guanine nucleotide exchange factor, are fundamental Gβγ effectors in the pathways controlling directionally persistent motility. In addition, GPCR-dependent chemotactic responses often involve endosomal trafficking of signaling proteins; coincidently, endosomes serve as signaling platforms for Gβγ In preclinical murine models of cancer, inhibition of Gβγ attenuates tumor growth, whereas in cancer patients, aberrant overexpression of chemotactic Gβγ effectors and recently identified mutations in Gβ correlate with poor clinical outcome. Here we discuss emerging paradigms of Gβγ signaling in cancer, which are essential for chemotactic cell migration and represent novel opportunities to develop pathway-specific pharmacologic treatments.

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G protein coupled receptor signaling complexes in live cells

Cited by 11 publications

References 33 publications

RGS14 regulates the lifetime of Gα‐GTP signaling but does not prolong Gβγ signaling following receptor activation in live cells

RGS14 regulates the lifetime of Gα‐GTP signaling but does not prolong Gβγ signaling following receptor activation in live cells

Distinct Signaling Patterns of Allosteric Antagonism at the P2Y₁ Receptor

Gβγ Pathways in Cell Polarity and Migration Linked to Oncogenic GPCR Signaling: Potential Relevance in Tumor Microenvironment

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G protein coupled receptor signaling complexes in live cells

Cited by 11 publications

References 33 publications

RGS14 regulates the lifetime of Gα‐GTP signaling but does not prolong Gβγ signaling following receptor activation in live cells

RGS14 regulates the lifetime of Gα‐GTP signaling but does not prolong Gβγ signaling following receptor activation in live cells

Distinct Signaling Patterns of Allosteric Antagonism at the P2Y1 Receptor

Gβγ Pathways in Cell Polarity and Migration Linked to Oncogenic GPCR Signaling: Potential Relevance in Tumor Microenvironment

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Distinct Signaling Patterns of Allosteric Antagonism at the P2Y₁ Receptor