A2B adenosine receptor activation and modulation by protein kinase C.

Protein kinase C (PKC) isoforms regulate many important signaling pathways. Here, we report that PKC activation by phorbol 12-myristate 13-acetate (PMA) enhanced A2B adenosine receptor (AR)-mediated, but not ß2-adrenergic receptor-mediated, cAMP accumulation, in H9C2 cardiomyocyte-like and HEK293 cells. In addition to enhancement, PKC (PMA-treatment) also activated A2BAR with low Emax (H9C2 and NIH3T3 cells endogenously expressing A2BAR), or with high Emax (A2BAR-overexpressing HEK293 cells) to induce cAMP accumulation. A2BAR activation induced by PKC was inhibited by A2BAR and PKC inhibitors but enhanced by A2BAR overexpression. Gαi isoforms and PKCγ isoform were found to be involved in both enhancement of A2BAR function and A2BAR activation. Thus, we establish PKC as an endogenous modulator and activator of A2BAR, involving Giα and PKCγ. Depending on signaling pathway, PKC could activate and enhance, or alternatively inhibit A2BAR activity. These findings are relevant to common functions of A2BAR and PKC, e.g. cardioprotection and cancer progression/treatment.

Patient-specific variants of NFU1/NFU-1 disrupt cholinergic signaling in a model of multiple mitochondrial dysfunctions syndrome 1.

Neuromuscular dysfunction is a common feature of mitochondrial diseases and frequently presents as ataxia, spasticity and/or dystonia, all of which can severely impact individuals with mitochondrial diseases. Dystonia is one of the most common symptoms of multiple mitochondrial dysfunctions syndrome 1 (MMDS1), a disease associated with mutations in the causative gene (NFU1) that impair iron-sulfur cluster biogenesis. We have generated Caenorhabditis elegans strains that recreated patient-specific point variants in the C. elegans ortholog (nfu-1) that result in allele-specific dysfunction. Each of these mutants, Gly147Arg and Gly166Cys, have altered acetylcholine signaling at neuromuscular junctions, but opposite effects on activity and motility. We found that the Gly147Arg variant was hypersensitive to acetylcholine and that knockdown of acetylcholine release rescued nearly all neuromuscular phenotypes of this variant. In contrast, we found that the Gly166Cys variant caused predominantly postsynaptic acetylcholine hypersensitivity due to an unclear mechanism. These results are important for understanding the neuromuscular conditions of MMDS1 patients and potential avenues for therapeutic intervention.

Noncanonical scaffolding of Gαi and ß-arrestin by G protein-coupled receptors.

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are common drug targets and canonically couple to specific Gα protein subtypes and ß-arrestin adaptor proteins. G protein-mediated signaling and ß-arrestin-mediated signaling have been considered separable. We show here that GPCRs promote a direct interaction between Gαi protein subtype family members and ß-arrestins regardless of their canonical Gα protein subtype coupling. Gαi:ß-arrestin complexes bound extracellular signal-regulated kinase (ERK), and their disruption impaired both ERK activation and cell migration, which is consistent with ß-arrestins requiring a functional interaction with Gαi for certain signaling events. These results introduce a GPCR signaling mechanism distinct from canonical G protein activation in which GPCRs cause the formation of Gαi:ß-arrestin signaling complexes.