Nuclear localisation and function of the g protein-coupled receptor kinase 4 subfamily.

Abstract

G protein-coupled receptor kinases (GRKs) are a family of serine/threonine kinases that phosphorylate and mediate densensitisation of agonist-occupied G protein-coupled receptors (GPCRs). Recent evidence suggests, however, additional cellular functions for these enzymes. The seven mammalian GRKs are divided into the GRK1-, GRK2- and GRK4-like subfamilies. I have discovered that GRK4 subfamily members contain functional nuclear localisation sequences (NLSs). The location of the NLSs of GRK5 and 6 is similar whereas the NLS of GRK4 is more N-terminal. Although all members of the GRK4 subfamily contain NLSs the nuclear localisation of these kinases is differentially regulated by Ca2+/CaM. Furthermore, the NLS of GRKs 5 and 6, but not GRK4, mediate DNA-binding in vitro, suggesting differential nuclear signalling roles for these enzymes. I investigated structural determinants of GRK5 required for DNA-binding and nuclear localisation. The ability of GRK5 to bind DNA requires an intact NLS and an N-terminally located calmodulin (CaM) binding domain. Additionally, CaM- dependent autophosphorylation of GRK5 reduces the affinity of GRK5 for DNA. Overexpression of GRK5 in cultured rat neonatal cardiac myocytes rescues hypertrophy induced by phenylephrine, an oc-adrenergic receptor agonist, or by activation of PLC. Hypertrophy is not rescued by over expression of GRK5 with a mutant NLS (GRK5ANLS), mutant N-terminal CaM binding site (GRK5NTPB) or a catalytically in active version of the kinase (GRK5K215R). That GRK5 and GRK5ANLS show similar kinase activity in vitro suggests that both the kinase activity and the DNA-binding ability of GRK5 contributes to its role in rescuing hypertrophy. Since GRK5 rescues hypertrophy induced downstream of adrenergic receptors, by PLC activation, GRK5 DNA-binding ability and kinase activity in the nucleus would appear to be required for this effect. Clearly, the GRK4 subfamily of GRKs is more diverse than previously appreciated and play a role in cellular signalling in the nucleus distinct from GPCR desensitisation