Control of Gα _q signaling dynamics and GPCR cross-talk by GRKs

Abstract

Numerous processes contribute to the regulation of G protein–coupled receptors (GPCRs), but relatively little is known about rapid mechanisms that control signaling on the seconds time scale or regulate cross-talk between receptors. Here, we reveal that the ability of some GPCR kinases (GRKs) to bind Gα q both drives acute signaling desensitization and regulates functional interactions between GPCRs. GRK2/3-mediated acute desensitization occurs within seconds, is rapidly reversible, and can occur upon local, subcellular activation. This rapid desensitization is kinase independent, insensitive to pharmacological inhibition, and generalizable across receptor families and effectors. We also find that the ability of GRK2 to bind G proteins also enables it to regulate the extent and timing of Gα q -dependent signaling cross-talk between GPCRs. Last, we find that G protein/GRK2 interactions enable a novel form of GPCR trafficking cross-talk. Together, this work reveals potent forms of Gα q -dependent GPCR regulation with wide-ranging pharmacological and physiological implications.

Synaptic plasticity via receptor tyrosine kinase/G-protein-coupled receptor crosstalk

“…We then co-expressed a membrane-tethered version of GPCR kinase 2 (“GRK2-CAAX”), which can bind and sequester both Gβγ and Gα q -GTP. 68 , 69 Indeed, GRK2-CAAX co-expression reduced the proportion of cells responding to BDNF with Ca 2+ oscillations ( Figure 5B ) without altering BDNF responses in cells expressing TrkB alone ( Figure S6C ) or mGluR5 glutamate responses ( Figure S6D ). Mutation of both the Gβγ (R587Q) and the Gα q (D110A) binding sites on GRK2 was required to rescue efficient TrkB/mGluR5 crosstalk ( Figure 5B ).…”

“… 4 , 80 mGluR1 co-expression allowed clear responses to BDNF in ~20%–40% of cells ( Figures 7C and S8B ), but 5-HT 2A R co-expression enabled responses in only ~5%–10% of cells ( Figure 7C ). Based on our prior work, 69 we reasoned that 5-HT 2A R may not produce sufficient tonic Gα q activation and added a subthreshold level of 5-HT (1 nM). This boosted the proportion of cells responding to BDNF to 20%–30% ( Figures 7B and 7C ).…”

Histamine H1 Receptor-Mediated JNK Phosphorylation Is Regulated by Gq Protein-Dependent but Arrestin-Independent Pathways

“…To investigate the effects of various inhibitors, CHO cells were treated with 100 µM histamine for 30 min in the presence and absence (vehicle) of the inhibitors. Inhibitors used were the following: histamine H 1 receptor antagonists, ketotifen (Sigma-Aldrich) and diphenhydramine (Sigma-Aldrich) [ 43 ]; a G q protein inhibitor, YM-254890 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) [ 48 ]; an intracellular Ca 2+ chelator, BAPTA-AM (Abcam, Cambridge, UK) [ 49 ]; a protein kinase C (PKC) inhibitor, GF109203X (Sigma-Aldrich) [ 50 ]; a GRK2/3 inhibitor, cmpd101 (Hello Bio, Bristol, UK) [ 51 ]; and a dynamin inhibitor, dynasore (Sigma-Aldrich) [ 52 ]. These test drugs were dissolved in 10% dimethyl sulfoxide (DMSO) (FUJIFILM Wako Pure Chemical Corporation) and the final concentration of DMSO in the reaction medium was 0.1%.…”

Distinct beta-arrestin coupling and intracellular trafficking of metabotropic glutamate receptor homo- and heterodimers

“…For the surface labeling internalization assay (Fig. 1G), we followed our previously described protocol ( 21 , 93 ). Twenty-four hours after transfection, HEK 293T cells expressing SNAP-tagged receptors (0.2 to 0.5 μg alone or in combination with untagged receptors) were washed with fresh media and incubated in saturating antagonist (negative control; 10 μM LY34 for mGluR2 or mGluR3, 20 μM CPPG for mGluR8, 10 μM naloxone for MOR, and no drug for V2R) or agonist (1 mM Glu, 10 μM DAMGO, and 100 nM vasopressin) in media for 1 hour at 37°C.…”