GRK2 Protein Research Articles

Differentially Regulated Expression of the G-Protein–Coupled Receptor Kinases, βARK and GRK6, During Myelomonocytic Cell Development In Vitro

G-protein-coupled receptor kinases (GRKs) mediate agonist-specific phosphorylation and desensitization of G-protein-coupled receptors. Previous studies have shown that several of these kinases are expressed in hematopoietic cells and that GRK expression is modulated during T-lymphocyte activation. Here, we analyzed the regulation of beta-adrenergic receptor kinase (betaARK) and GRK6 expression and activity in myelomonocytic and lymphoid cells. In the promyelocytic cell line HL-60, GRK6 protein levels and activity rose twofold to fourfold over the course of treatment with agents that induce differentiation toward either the myeloid (dimethyl sulfoxide and retinoic acid) or monocytic [1alpha,25(OH)2-vitamin D3] lineage, whereas betaARK protein levels and activity were only slightly altered. In contrast, treatment with phorbol 12,13-myristic acetate (PMA) led to a reduction in steady-state levels and activity of both betaARK and GRK6. Treatment of human lymphocytes with several polyclonal activators (phytohemagglutinin, anti-CD3 antibody and interleukin-2) resulted in enhanced betaARK and GRK6 mRNA and protein levels and increased activity of both kinases. In contrast, PMA and calcium ionophore treatment significantly elevated GRK6 protein levels, while decreasing betaARK expression. These data demonstrate that betaARK and GRK6 expression are differentially regulated during myelomonocytic cell development and lymphocyte activation.

Expression and regulation of G protein-coupled receptor kinase 5 and beta-arrestin-1 in rat thyroid FRTL5 cells.

G protein-coupled receptor kinases (GRKs) and arrestins are implicated in homologous desensitization of G protein-coupled receptors. We have recently demonstrated that among six GRKs so far identified, GRK5 is the isoform predominantly expressed in the thyroid and appears to be mainly involved in homologous desensitization of thyrotropin receptor (TSHR) in FRTL5 cells. To further understand the molecular mechanisms of the TSHR desensitization, the expression and regulation of GRKs and arrestins together with those of the TSHR were examined in FRTL5 cells. Northern blot analysis of total RNA from FRTL5 cells with the available rat GRK cDNAs (GRK4, 5, and 6) as probes showed that only GRK5 mRNAs of approximately 3, 8, and 10 kilo bases (kb) in length were detectable. When probed with rat beta-arrestin-1 and beta-arrestin-2 cDNAs, beta-arrestin-1 mRNAs of approximately 7.5 and 2.5 kb long, but no (or possibly faint) approximately 2.4 kb beta-arrestin-2 mRNA, were observed, suggesting that in the thyroid, beta-arrestins appear to be predominantly of beta-arrestin-1 isoform. In studies on TSH-regulation of GRK5, beta-arrestin-1 and TSHR mRNAs, steady-state levels of GRK5 and TSHR mRNAs were 3- to 4-fold lower in the cells grown in the medium with TSH than in those without TSH, while betaarrestin-1 mRNA levels were unchanged. Downregulation of GRK5 and TSHR mRNAs by TSH was further confirmed by dose- and time-dependent experiments. Incubation with 1mM 8BrcAMP, a cAMP analog, for 24h fully reproduced this TSH inhibitory effect. A decrease in GRK5 protein by TSH was also confirmed with Western blot analysis. In summary, these data together with our previous data suggested that GRK5 and beta-arrestin-1 seem to be the isoforms predominantly expressed in the thyroid, and they appear to play a pivotal role in TSHR homologous desensitization. We also demonstrated TSH downregulation of GRKS, but not beta-arrestin-1, expression. Further studies will be necessary to elucidate how these phenomena are linked to thyroid pathophysiology.

Identification, purification, and characterization of GRK5, a member of the family of G protein-coupled receptor kinases.

A novel member of the family of G protein-coupled receptor kinases (GRKs), named GRK5, has been cloned from bovine taste epithelium. The cDNA sequence predicts a 590-amino acid protein with high overall similarity to rhodopsin kinase. GRK5 mRNA is found most abundantly in lung, heart, retina, and lingual epithelium, but is expressed very little in brain, liver, kidney, or testis. GRK5 expressed in Sf9 cells was purified to apparent homogeneity. GRK5 major autophosphorylation sites were mapped to Ser484 and Thr485. Purified GRK5 phosphorylates rhodopsin in a light-dependent manner and beta 2-adrenergic receptor in an agonist-dependent manner and phosphorylates the C-terminal tail regions of both receptor proteins. GRK5 possesses neither a CAAX motif specifying protein prenylation like rhodopsin kinase nor similarity to the G protein beta gamma-subunit binding domain of beta-adrenergic receptor kinases. GRK5 phosphorylation of rhodopsin or beta 2-adrenergic receptor is not stimulated by G protein beta gamma-subunits. The GRK5 protein does not undergo agonist-dependent translocation from cytosol to membranes as do beta-adrenergic receptor kinase and rhodopsin kinase, but rather appears to associate with membranes constitutively. GRK5 thus appears functionally similar to other characterized GRKs, but has distinct regulatory properties which may be important for its cellular function.