Expression and Regulation of G Protein-Coupled Receptor Kinase 5 and β-Arrestin-1 in Rat Thyroid FRTL5 Cells

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Abstract
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.