Upon thyrotropin binding the thyrotropin receptor is internalized and localized to endosome.
To study the fate of TSH receptor (TSHR) on TSH binding, we constructed a chimeric cDNA that encodes a yellow fluorescent protein (YFP) fused to the carboxyl terminus of human TSHR. The protein expression in transfected cells was confirmed using flow cytometry. The functionality of the chimeric protein was determined by its ability to transduce signal leading to activation of cAMP in a TSH dose-dependent manner. The levels of cAMP produced by these cells were comparable with the levels seen in cells transfected with unfused TSHR without the YFP. Using deconvolution microscopy, we observed that the receptor is largely expressed on the cell surface, but on addition of TSH, some of the receptors were rapidly internalized. This conclusion was supported by several independent observations involving different cells expressing either native or recombinant TSHR. On TSH treatment, we observed internalization of human TSHR-YFP and human TSHR, expressed on 293 and CHO cells, respectively. This was further substantiated when we observed colocalization of rhodamine-labeled TSH with TSHR-YFP within the cell and by the uptake of radiolabeled TSH. Furthermore, shortly after ligand binding, there was a profound change in the morphology of the cells and some of the receptors accumulated in the perinuclear region of the cell. The TSHR-YFP was colocalized with RhoB-cyan fluorescent protein, indicating that it accumulated within the endosomes. These results indicate that the receptor internalization might in part be responsible for TSHR desensitization on TSH binding.
Department of Microbiology and Immunology (M/C 790), College of Medicine, University of Illinois at Chicago, Room E-705, 835 South Wolcott Avenue, Chicago, IL 60612, USA.
Surya P
SP
Singh
David
D
McDonald
Thomas J
TJ
Hope
Bellur S
BS
Prabhakar
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D018076:DNA, Complementary / Q000235:genetics; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D011989:Receptors, Thyrotropin / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013972:Thyrotropin / Q000378:metabolism*; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D013972:Thyrotropin; D000242:Cyclic AMP