Abstract
The G protein-coupled receptor kinase (GRK2) belongs to a family of protein kinases that phosphorylates agonist-activated G protein-coupled receptors, leading to G protein-receptor uncoupling and termination of G protein signaling. GRK2 also contains a regulator of G protein signaling homology (RH) domain, which selectively interacts with α-subunits of the Gq/11 family that are released during G protein-coupled receptor activation. We have previously reported that kinase activity of GRK2 up-regulates activity of the epithelial sodium channel (ENaC) in a Na(+) absorptive epithelium by blocking Nedd4-2-dependent inhibition of ENaC. In the present study, we report that GRK2 also regulates ENaC by a mechanism that does not depend on its kinase activity. We show that a wild-type GRK2 (wtGRK2) and a kinase-dead GRK2 mutant ((K220R)GRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (ΔRH-GRK2) or a GRK2 mutant that cannot interact with Gαq/11/14 ((D110A)GRK2), increase activity of ENaC. GRK2 up-regulates the basal activity of the channel as a consequence of its RH domain binding the α-subunits of Gq/11. We further found that expression of constitutively active Gαq/11 mutants significantly inhibits activity of ENaC. Conversely, co-expression of siRNA against Gαq/11 increases ENaC activity. The effect of Gαq on ENaC activity is not due to change in ENaC membrane expression and is independent of Nedd4-2. These findings reveal a novel mechanism by which GRK2 and Gq/11 α-subunits regulate the activity ENaC.
Highlights
The -adrenergic receptor kinase 1 (GRK2) is one of the G protein-coupled receptor serine/threonine kinases (GRKs).3 All seven members of the GRK family (GRK1–7) share a highly homologous kinase domain that is flanked toward the C-terminal by a pleckstrin homology (PH) domain and toward the N-terminal by a regulator of G-protein signaling homology (RH) domain [1]
We show that a wild-type GRK2 and a kinase-dead GRK2 mutant (K220RGRK2), but not a GRK2 mutant that lacks the C-terminal RH domain (⌬RHGRK2) or a GRK2 mutant that cannot interact with G␣q/11/14 (D110AGRK2), increase activity of epithelial Naϩ channels (ENaC)
We found that this stimulatory effect of GRK2 on ENaC can be attributed to the ability of the RH domain of GRK2 to interact with and inhibit the ␣-subunit of Gq/11, which acts as a negative regulator of ENaC
Summary
The -adrenergic receptor kinase 1 (GRK2) is one of the G protein-coupled receptor serine/threonine kinases (GRKs).3 All seven members of the GRK family (GRK1–7) share a highly homologous kinase domain that is flanked toward the C-terminal by a pleckstrin homology (PH) domain and toward the N-terminal by a regulator of G-protein signaling homology (RH) domain [1]. We found that over-expression of GRK2 significantly increased the normalized amiloride-sensitive Naϩ current (1.53 Ϯ 0.17, n ϭ 6 versus 1.00 Ϯ 0.07, n ϭ 6 in untransfected cells; p Ͻ 0.05), consistent with GRK2 positively regulating the activity of ENaC
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