The Human Thyrotropin Receptor Is Predominantly Internalized by β-Arrestin 2

Abstract

The beta-arrestin-dependent endocytosis of the beta2-adrenergic receptor (beta2AR) has been demonstrated by confocal fluorescence microscopy. Furthermore, a constitutively activated beta2AR is also constitutively desensitized and down-regulated. To clarify the function of beta-arrestin 1 or 2 for TSH receptor (TSHR) desensitization and examine whether constitutively activated TSHR mutants are internalized in a different way, we investigated the TSHR trafficking in association with beta-arrestins in cotransfection experiments in HEK 293 cells using confocal laser-scanning microscopy. We found that both beta-arrestins are able to internalize the TSHR in HEK 293 cells. However, whereas the beta-arrestin 1-mediated TSHR internalization reached its maximum 20 min after TSH stimulation, the beta-arrestin 2-mediated TSHR internalization already reached its maximum 5 min after TSH stimulation. Furthermore, an increased basal desensitization and internalization of constitutively activated TSHR mutants N670S, S505N, and F631L cotransfected with beta-arrestin 2 could not be found. After TSH stimulation the constitutively activated mutants showed the same time course for internalization as the wild-type-TSHR. In summary, contrary to data obtained for the beta2AR, the constitutive activation of the TSHR does not influence the desensitization and time course for internalization of the receptor, and in agreement with findings for the FSH and LH receptors, these results characterize the TSH receptor as a member of the class A of G protein-coupled receptors, which have a higher affinity to beta-arrestin 2 than beta-arrestin 1 and do not colocalize with beta-arrestins in endosomes.

Thyrotropin Receptor Stimulates Internalization-Independent Persistent Phosphoinositide Signaling

“…8). There was no apparent TSHR internalization in HEK-TSHR cells or in HEK-TSHR cells expressing K44A (data not shown) but robust internalization in HEK-TSHR cells expressing ␤Arr2, as has been reported previously (Frenzel et al, 2006;Neumann et al, 2010a). However, G␣ q -YFP remained at the surface of all cells including cells expressing ␤Arr2 (Fig.…”

Characterization of Relaxin Receptor (RXFP1) Desensitization and Internalization in Primary Human Decidual Cells and RXFP1-Transfected HEK293 Cells

“…Desensitization, internalization, and functional importance of the arrestin proteins have been demonstrated for other LGR GPCRs: TSHR, LHR, and FSHR. These receptors undergo ␤-arrestin 1 and 2-dependent desensitization with enhanced internalization by ␤-arrestin 2 (20 -22), TSHR has a higher affinity to ␤-arrestin 2 than ␤-arrestin 1 (22). We investigated the desensitization and down-regulation of signaling of RXFP1 after relaxin treatment.…”

Normal testicular function without detectable follicle-stimulating hormone. A novel mutation in the follicle-stimulating hormone receptor gene leading to apparent constitutive activity and impaired agonist-induced desensitization and internalization

“…Since constitutively active mutant GPCRs may or may not exhibit constitutive desensitization and down-regulation, depending on the particular receptor and the location of the activating mutation (Frenzel et al, 2006;Pei et al, 1994;Quellari et al, 2003), we studied the kinetics of desensitization and internalization of the N431I mutant FSHR. For this purpose, HEK-293 transiently transfected with the WT and mutant FSHR cDNAs were incubated with increasing concentrations of FSH for 2 h and then exposed to high concentrations of agonist for 30 min.…”