J L Benovic

Multiple mechanisms involving protein phosphorylation are linked to desensitization of muscarinic receptors.

Agonists induce phosphorylation of m2 muscarinic receptors (mAChR) in several cell types. This phosphorylation correlates with desensitization. The mechanisms underlying mAChR phosphorylation have been investigated using several in vitro approaches. Protein kinase C phosphorylated the purified and reconstituted m2 mAChR to a stoichiometry of approximately 5 mols P/mol receptor; this phosphorylation resulted in the decreased ability of receptors to activate G-proteins. Although the phosphorylation by PKC was not modulated by agonist binding to the mAChR, heterotrimeric G-proteins were able to completely block the PKC-mediated effects. If significant receptor/G-protein coupling occurs in vivo, agonists would be required to promote dissociation of the G-proteins from the receptors and reveal the phosphorylation sites for PKC. Members of the G-protein coupled receptor kinase (GRK) family also phosphorylated the purified and reconstituted m2 mAChR. In contrast to PKC, the GRKs phosphorylated the m2 mAChR strictly in an agonist-dependent manner. GRK mediated phosphorylation perturbed receptor/G-protein coupling. In addition, phosphorylation allowed for arrestin binding to the m2 mAChR which should further contribute to desensitization. Using a new strategy that does not require purification and reconstitution of receptors for GRK studies, the m3 mAChR were revealed as substrates for the GRKs. For both the m2 and m3 receptor subtypes, the most effective kinases were GRK 2 and 3. Phosphorylation of the receptors by these enzymes was stimulated by low concentrations of G-proteins and by membrane phospholipids. Thus, multiple mechanisms involving protein phosphorylation appear to contribute to the overall process of mAChR desensitization.

Caspase-cleaved arrestin-2 and BID cooperatively facilitate cytochrome C release and cell death

Apoptosis is programmed cell death triggered by activation of death receptors or cellular stress. Activation of caspases is the hallmark of apoptosis. Arrestins are best known for their role in homologous desensitization of G protein-coupled receptors (GPCRs). Arrestins quench G protein activation by binding to activated phosphorylated GPCRs. Recently, arrestins have been shown to regulate multiple signalling pathways in G protein-independent manner via scaffolding signalling proteins. Here we demonstrate that arrestin-2 isoform is cleaved by caspases during apoptosis induced via death receptor activation or by DNA damage at evolutionarily conserved sites in the C-terminus. Caspase-generated arrestin-2-(1-380) fragment translocates to mitochondria increasing cytochrome C release, which is the key checkpoint in cell death. Cells lacking arrestin-2 are significantly more resistant to apoptosis. The expression of wild-type arrestin-2 or its cleavage product arrestin-2-(1-380), but not of its caspase-resistant mutant, restores cell sensitivity to apoptotic stimuli. Arrestin-2-(1-380) action depends on tBID: at physiological concentrations, arrestin-2-(1-380) directly binds tBID and doubles tBID-induced cytochrome C release from isolated mitochondria. Arrestin-2-(1-380) does not facilitate apoptosis in BID knockout cells, whereas its ability to increase caspase-3 activity and facilitate cytochrome C release is rescued when BID expression is restored. Thus, arrestin-2-(1-380) cooperates with another product of caspase activity, tBID, and their concerted action significantly contributes to cell death.

Molecular events associated with the regulation of signaling by M2 muscarinic receptors.

Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs.