1. |
- Ham, Seungmin, et al.
(författare)
-
Role of G protein-coupled receptor kinases (GRKs) in β2-adrenoceptor-mediated glucose uptake
- 2024
-
Ingår i: Pharmacology Research & Perspectives. - 2052-1707. ; 12:1
-
Tidskriftsartikel (refereegranskat)abstract
- Truncation of the C-terminal tail of the beta(2)-AR, transfection of beta ARKct or over-expression of a kinase-dead GRK mutant reduces isoprenaline-stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the beta(2)-AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO-GLUT4myc cells expressing wild-type and mutant beta(2)-ARs were generated and receptor affinity for [H-3]-CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by beta(2)-AR agonists, cAMP accumulation, GLUT4 translocation, [H-3]-2-deoxyglucose uptake, and beta(2)-AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between beta(2)-AR and beta-arrestin2 or between beta(2)-AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to beta(2)-AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to beta(2)-AR agonists occurred in CHO-GLUT4myc cells expressing beta(2)-ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild-type beta(2)-AR. However, beta(2)-ARs lacking phosphorylation sites failed to recruit beta-arrestin2 and did not internalize. GRK2 knock-down or GRK2 inhibitors decreased isoprenaline-stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the beta(2)-AR is not associated with isoprenaline- or BRL37344-stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock-down or GRK2 inhibition reduces isoprenaline-stimulated glucose uptake.
|
|
2. |
- Sato, Masaaki, et al.
(författare)
-
Interaction with Caveolin-1 Modulates G Protein Coupling of Mouse beta(3)-Adrenoceptor
- 2012
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:24, s. 20674-20688
-
Tidskriftsartikel (refereegranskat)abstract
- Caveolins act as scaffold proteins in multiprotein complexes and have been implicated in signaling by G protein-coupled receptors. Studies using knock-out mice suggest that beta(3)-adrenoceptor (beta(3)-AR) signaling is dependent on caveolin-1; however, it is not known whether caveolin-1 is associated with the beta(3)-AR or solely with downstream signaling proteins. We have addressed this question by examining the impact of membrane rafts and caveolin-1 on the differential signaling of mouse beta(3a)- and beta(3b)-AR isoforms that diverge at the distal C terminus. Only the beta(3b)-AR promotes pertussis toxin (PTX)-sensitive cAMP accumulation. When cells expressing the beta(3a)-AR were treated with filipin III to disrupt membrane rafts or transfected with caveolin-1 siRNA, the cyclic AMP response to the beta(3)-AR agonist CL316243 became PTX-sensitive, suggesting G alpha(i/o) coupling. The beta(3a)-AR C terminus, S (P-384) under bar PLNR (P-389) under bar DG (Y-392) under bar EGARP (P-398) under bar PT, resembles a caveolin interaction motif. Mutant beta(3a)-ARs (F389A/Y392A/F398A or P384S/F389A) promoted PTX-sensitive cAMP responses, and in situ proximity assays demonstrated an association between caveolin-1 and the wild type beta(3a)-AR but not the mutant receptors. In membrane preparations, the beta(3b)-AR activated G alpha(o) and mediated PTX-sensitive cAMP responses, whereas the beta(3a)-AR did not activate G alpha(i/o) proteins. The endogenous beta(3a)-AR displayed G alpha(i/o) coupling in brown adipocytes from caveolin-1 knock-out mice or in wild type adipocytes treated with filipin III. Our studies indicate that interaction of the beta(3a)-AR with caveolin inhibits coupling to G alpha(i/o) proteins and suggest that signaling is modulated by a raft-enriched complex containing the beta(3a)-AR, caveolin-1, G alpha(s), and adenylyl cyclase.
|
|