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 β(3)-adrenoceptor (β(3)-AR) signaling is dependent on caveolin-1; however, it is not known whether caveolin-1 is associated with the β(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 β(3a)- and β(3b)-AR isoforms that diverge at the distal C terminus. Only the β(3b)-AR promotes pertussis toxin (PTX)-sensitive cAMP accumulation. When cells expressing the β(3a)-AR were treated with filipin III to disrupt membrane rafts or transfected with caveolin-1 siRNA, the cyclic AMP response to the β(3)-AR agonist CL316243 became PTX-sensitive, suggesting Gα(i/o) coupling. The β(3a)-AR C terminus, SP(384)PLNRF(389)DGY(392)EGARPF(398)PT, resembles a caveolin interaction motif. Mutant β(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 β(3a)-AR but not the mutant receptors. In membrane preparations, the β(3b)-AR activated Gα(o) and mediated PTX-sensitive cAMP responses, whereas the β(3a)-AR did not activate Gα(i/o) proteins. The endogenous β(3a)-AR displayed Gα(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 β(3a)-AR with caveolin inhibits coupling to Gα(i/o) proteins and suggest that signaling is modulated by a raft-enriched complex containing the β(3a)-AR, caveolin-1, Gα(s), and adenylyl cyclase.
Highlights
Caveolins affect signaling by G protein-coupled receptors (GPCRs)
We proposed that binding of proteins such as caveolin or other scaffolding proteins to the 3a-AR C terminus may localize the receptor to membrane microdomains or intracellular compartments where it cannot couple to guanine nucleotidebinding proteins (G␣i/o)
We examined the properties of the wild type 3a-AR, 3b-AR, and a truncated 3-AR that lack the C-terminal tail of either splice variant in clonal CHO-K1 cell lines with equivalent receptor densities (Bmax 1148 Ϯ 241, 1309 Ϯ 128, and 1224 Ϯ 105 fmol/mg protein, respectively)
Summary
Caveolins affect signaling by G protein-coupled receptors (GPCRs). Results: Interaction between 3a-adrenoceptors and caveolin-1 facilitates Gs-mediated responses but prevents the receptor from coupling to inhibitory Gi/o proteins. No studies to date have reported localization of the 3-AR in membrane rafts or caveolae, there is firm evidence that caveolin-1 regulates 3-AR signaling in adipocytes In both white and brown adipocytes, 3-ARs stimulate the G␣s/ adenylyl cyclase/protein kinase A (PKA) pathway, promoting breakdown of fat (lipolysis) via phosphorylation of perilipin and hormone-sensitive lipase. There is no change in the PTX sensitivity of the 3b-AR response This suggests that residues present in the 3a-AR C-terminal tail may direct localization of the receptor to membrane rafts, and this in turn may govern its capacity to couple to G␣i/o proteins. PTX treatment increases cAMP responses in membranes derived from cells expressing the 3b-AR via inhibition of receptor-G␣o coupling
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