Heterotrimeric G Protein-independent Signaling of a G Protein-coupled Receptor

Self Cite

The A 2A -adenosine receptor is a prototypical G s protein-coupled receptor but stimulates MAPK/ERK in a G s -independent way. The A 2A receptor has long been known to undergo restricted collision coupling with G s ; the mechanistic basis for this mode of coupling has remained elusive. Here we visualized agonist-induced changes in mobility of the yellow fluorescent protein-tagged receptor by fluorescence recovery after photobleaching microscopy. Stimulation with a specific A 2A receptor agonist did not affect receptor mobility. In contrast, stimulation with dopamine decreased the mobility of the D 2 receptor. When coexpressed in the same cell, the A 2A receptor precluded the agonist-induced change in D 2 receptor mobility. Thus, the A 2A receptor did not only undergo restricted collision coupling, but it also restricted the mobility of the D 2 receptor. Restricted mobility was not due to tethering to the actin cytoskeleton but was, in part, related to the cholesterol content of the membrane. Depletion of cholesterol increased receptor mobility but blunted activation of adenylyl cyclase, which was accounted for by impaired formation of the ternary complex of agonist, receptor, and G protein. These observations support the conclusion that the A 2A receptor engages G s and thus signals to adenylyl cyclase in cholesterol-rich domains of the membrane. In contrast, stimulation of MAPK by the A 2A receptor was not impaired. These findings are consistent with a model where the recruitment of these two pathways occurs in physically segregated membrane microdomains. Thus, the A 2A receptor is the first example of a G protein-coupled receptor documented to select signaling pathways in a manner dependent on the lipid microenvironment of the membrane.In the fluid mosaic model, the lipid bilayer is an isotropic milieu, in which membrane-embedded proteins diffuse in two dimensions and thus collide at random with each other (1). When applied to G protein-coupled receptors, the model predicts that G protein-coupled receptors move in a random walk, and, upon activation, this allows them to engage their cognate G proteins. This "collision coupling" mode of activation was validated in studies using the ␤-adrenergic receptor and its coupling to the effector enzyme adenylyl cyclase in turkey erythrocytes (2). However, experiments with the A 2 -adenosine receptor in turkey erythrocytes revealed kinetic properties of adenylyl cyclase activation that were incompatible with the collision coupling model. The results indicated a tight coupling of the adenosine receptor to G␣ s (3, 4); the term "restricted collision coupling" was coined to account for this altered mode of coupling. Restricted collision is not a feature unique to the avian A 2 -adenosine receptor, because it was also documented for the human A 2A receptor in platelet membranes (5). In addition, the A 2A -adenosine receptor has the unusual feature of forming a tight complex with G s , which persists in detergent solution, which is resistant to guanine nucleotides and which re...

Section: Discussionsupporting

confidence: 81%

Section: Lateral Mobility Of the A 2a Receptor Is Not Affected By Agomentioning

confidence: 99%

Restricted Collision Coupling of the A2A Receptor Revisited

Charalambous

Gsandtner

Keuerleber

et al. 2008

Self Cite

“…There are several A2AR-interacting proteins that may regulate the trafficking (48,(57)(58)(59), but to our knowledge only E3KARP and Ezrin are reported to interact with A2BR (29). However, the precise molecular mechanisms that regulate A2BR trafficking and function are still uncertain.…”

Section: Discussionmentioning

confidence: 99%

Adenosine A2A Receptor Is Involved in Cell Surface Expression of A2B Receptor

Moriyama

Sitkovsky

2010

The A2A and A2B adenosine receptors (A2AR and A2BR) are implicated in many physiological processes. However, the mechanisms of their intracellular maturation and trafficking are poorly understood. In comparative studies of A2AR versus A2BR expression in transfected cells, we noticed that the levels of cell surface expression of A2BR were significantly lower than those of A2AR. A large portion of the A2BR was degraded by the proteasome. Studies of cell surface expression of A2BR chimeric molecules in transfectants suggested that A2BR does not have the dominant forward transport signal for export from the endoplasmic reticulum to the cell surface. A2BR surface expression was increased in A2BR chimeras where the A2BR carboxyl terminus (CT) was replaced or fused with the A2AR CT. Co-transfection of A2AR with A2BR enhanced surface expression of A2BR though the F(X) 6 LL motif in the A2AR CT. The requirements of A2AR expression for better A2BR cell surface expression was not only established in transfectants but also confirmed by observations of much lower levels of A2BR-induced intracellular cAMP accumulation in response to A2BR-activating ligand in splenocytes from A2AR ؊/؊ mice than in wild type mice. The results of mechanistic studies suggested that poor A2BR expression at the cell surface might be accounted for mainly by the lack of a dominant forward transport signal from the endoplasmic reticulum to the plasma membrane; it is likely that A2BR forms a heterooligomer complex for better function.Newly synthesized proteins destined for secretory pathway have to translocate to the endoplasmic reticulum (ER) 2 for correct folding, assembly, and transport to the final destination site of action. It is believed that the newly synthesized proteins are monitored by an ER quality control system to avoid accumulation of incorrectly unfolded and unassembled proteins that may disrupt proper cellular processes (1-3).

“…In the third series of experiments, we determined whether ARF1-mediated ERK1/2 activation could be modulated by transient expression of ARNO and ARFGAP1, which facilitate GDP-GTP exchange and GTP hydrolysis of ARF1, respectively [43][44][45]. Expression of ARNO further augmented ERK1/2 activation by ARF1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These data indicate that the active from of ARF1 is a potent activator of the Raf1-MEK1-ERK1/2 pathway. It is interesting to note that ARNO is required for the sustained activation of ERK1/2 by the A2A adenosine receptor (43). Whether or not ARNO is also involved in the regulation of ␣ 2B -AR/ARF1-mediated ERK1/2 activation is under investigation.…”

Section: Discussionmentioning

confidence: 99%

Regulation of α2B-Adrenergic Receptor-mediated Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Activation by ADP-ribosylation Factor 1

Dong

2011

Background: ERK1/2 activation by G protein-coupled receptors can be mediated through multiple pathways. Results: ␣ 2B -Adrenergic receptor interacts with ADP-ribosylation factor 1 (ARF1) to modulate ERK1/2 activation. Conclusion: ERK1/2 activation by ␣ 2B -adrenergic receptor is mediated through a novel pathway involving ARF1. Significance: The small GTPase ARF1 may function as an important regulator in receptor-mediated ERK1/2 activation.