Adenylate Cyclase/cAMP/Protein Kinase A Signaling Pathway Inhibits Endothelin Type A Receptor-Operated Ca²⁺ Entry Mediated via Transient Receptor Potential Canonical 6 Channels

Abstract

Receptor-operated Ca 2ϩ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca 2ϩ concentration triggered by the activation of G q protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of G q protein-coupled endothelin type A receptor (ET A R)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ET A R and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca 2ϩ measurements with thapsigargin-induced Ca 2ϩ -depletion/Ca 2ϩ -restoration protocol to estimate ROCE showed that the stimulation of ET A R induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosinecAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6 S28A ) but not Thr69 (TRPC6 T69A ) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6 S28A and TRPC6 T69A ) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.

Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2)

“…In contrast, in pituitary cells, nonspecific cation channels, probably including TRPC6, are involved in PKA-stimulated calcium influx (106). PKA has been reported to phosphorylate mouse TRPC6 at two sites, Ser-28 and Thr-69 (corresponding to Thr-70 in humans), leading to channel inhibition (86,100). However, in one study, Ser-28 was felt to be the critical inhibitory site (100), whereas the other study noted that phosphorylation of Thr-69 was key (98).…”

“…9, A and C). In addition, abolishing the PKA/PKG phosphorylation site on Thr-70 (86,100) or the dual tyrosine phosphorylation sites at Tyr-31 and Tyr-285 (8) failed to significantly alter ERK activation by TRPC6 R895C (Fig. 9, A and C).…”

Agonist-promoted Ubiquitination Differentially Regulates Receptor Trafficking of Endothelin Type A and Type B Receptors

“…Generation of Stable Cell Lines-HEK293T cells stably expressing wild type or mutant N-terminally HA-tagged ETRs, and HEK293T cells stably expressing Myc-ubiquitin were generated by retroviral gene transfer as described previously (23,24). The positive cells were selected in DMEM containing 2 g/ml puromycin for a week, and individual lines were tested by Western blot using an anti-HA antibody.…”

Endothelin Receptor Signaling: New Insight Into Its Regulatory Mechanisms

“…Increasing evidence now suggests that TRPC channels are responsible for SOCE and ROCE (9,10,14,53). The TRPC family consisting of TRPC1-7 is divided phylogenetically into four distinct subfamilies (TRPC1; TRPC2; TRPC3, 6 and 7; TRPC4 and 5) (54).…”