α(2A)-Adrenergic receptor stimulated calcium release is transduced by G(i)-associated G(βγ)-mediated activation of phospholipase C

Proposed mechanisms by which α₂-adrenergic receptors (α₂AR) regulate intracellular calcium ([Ca²⁺](i)) include stimulation and inhibition of cell surface calcium channels, stimulation of calcium release via receptor coupling to G(q) with subsequent activation of phospholipase C and release of IP₃, or stimulation of calcium release via coupling to G(i) in an IP₃- independent manner. These potential mechanisms were explored in cells that expressed α(₂A)R endogenously (HEL cells), permanently transfected CHO cells, and transiently transfected COS-7 cells. Each cell type displayed agonist (UK14304)-dependent increases in [Ca²⁺](i) that were blocked by yohimbine, ablated by pertussis toxin, and largely unaffected by chelation of extracellular calcium. Furthermore, calcium release was associated with IP₃ accumulation and was blocked by an inhibitor of phospholipase C (PLC). When expressed in CHO cells, a mutated α(₂A)AR which has the amino and carboxyl termini of the third intracellular loop substituted with β₂AR sequence poorly coupled to G(i) in adenylyl cyclase assays, and likewise displayed virtually no coupling to increased [Ca²⁺](i). These results all point toward a G(i)- versus a G(q)-mediated coupling pathway triggering release of intracellular calcium stores. The possibility that G(βγ) subunits released from α(₂A)AR-G(i) coupling is the mechanism of PLC activation was explored in COS-7 cells by coexpressing α(2A)AR with the G(βγ) inhibitors transducin or a carboxy-terminal portion of the βAR kinase. Both βγ inhibitors markedly inhibited α(₂A)AR modulation of [Ca²⁺](i) while not affecting thromboxane A₂ receptor mediated stimulation of [Ca²⁺](i) via G(q) coupling. Thus, α(2A)AR couple to calcium release via G(i)-associated G(βγ) subunits. This coupling is present in multiple cell types and should be considered a major signal transduction pathway of this receptor.