Biomedical subjects

Chun-Rong Chen

Publications and source records attributed to Chun-Rong Chen.

21 records · Page 2Linked to original sources

PubMed2003

Evidence that the C terminus of the A subunit suppresses thyrotropin receptor constitutive activity.

The TSH receptor (TSHR), unlike the LH receptor (LHR), has considerable ligand-independent adenylyl cyclase activity, a feature of pathophysiological importance. The TSHR ectodomain partially suppresses constitutive activity, an effect reversed by trypsin treatment of intact cells. Localizing the functional site of trypsin action would provide insight into how the TSHR ectodomain exerts its constraint. For this purpose, we examined the effect of trypsin on intact cells expressing a series of modified TSHR. Trypsin did not increase cAMP production by a chimeric TSH-LH receptor involving substitution of TSHR residues 261-418 (the ectodomain C terminus). In contrast, with the wild-type TSHR, trypsin enhanced constitutive activity despite mutation of the following potential tryptic cleavage sites [arginine (R) and lysine (K) residues]: 1) K565, K651, K660 in the extracellular loops of the serpentine region; 2) B subunit juxtamembrane residues K371, K401, K415; 3) A subunit residues R310, R312, K313. We previously excluded K337 and K339 from being implicated in TSHR tryptic activation. By exclusion, only one R/K cluster remains as a possible target for the functional effect of trypsin, namely K287, K290, K291, and R293. Mutation of this cluster is incompatible with TSHR cell surface expression. However, tryptic clipping at this locus would reproduce a previously demonstrated structural effect of trypsin on the TSHR, removal of about a 2-kDa polypeptide fragment extending downstream from the locus to the C terminus of the A subunit. Taken together, these data suggest that the C terminus of the A subunit functions as a suppressor of TSHR constitutive activity.

Animals↗

PubMed2002

Thyroid-stimulating autoantibodies in Graves disease preferentially recognize the free A subunit, not the thyrotropin holoreceptor.

Graves disease is directly caused by thyroid-stimulating autoantibodies (TSAb's) that activate the thyrotropin receptor (TSHR). We observed upon flow cytometry using intact cells that a mouse mAb (3BD10) recognized the TSHR ectodomain with a glycosidylphosphatidylinositol (ECD-GPI) anchor approximately tenfold better than the same ectodomain on the wild-type TSHR, despite the far higher level of expression of the latter. The 3BD10 epitope contains the N-terminal cysteine cluster critical for TSAb action. Consequently, we hypothesized and confirmed that TSAb (but not thyrotropin-blocking autoantibodies [TBAb's]) also poorly recognize the wild-type TSHR relative to the ECD-GPI. Despite poor recognition by TSAb of the holoreceptor, soluble TSHR A subunits (known to be shed from surface TSHR) fully neutralized autoantibody-binding activity. These data indicate that the epitope(s) for TSAb's, but not for TBAb's, are partially sterically hindered on the holoreceptor by the plasma membrane, the serpentine region of the TSHR, or by TSHR dimerization. However, the TSAb epitope on the soluble A subunit is freely accessible. This observation, as well as other evidence, supports the concept that A subunit shedding either initiates or amplifies the autoimmune response to the TSHR, thereby causing Graves disease in genetically susceptible individuals.

Animals↗

PubMed2002

Immune deviation away from Th1 in interferon-gamma knockout mice does not enhance TSH receptor antibody production after naked DNA vaccination.

TSH receptor (TSHR) DNA vaccination induces high TSHR antibody levels in BALB/c mice housed in a conventional facility. However, under pathogen-free conditions, we observed a Th1 cellular response to TSHR antigen characterized by interferon-gamma (IFN gamma) production. In the present study we investigated the effect on TSHR DNA vaccination of diverting the cytokine milieu away from Th1 using 1) IFN gamma knockout BALB/c mice, and 2) wild-type mice covaccinated with DNA for the TSHR and for IFN gamma/receptor-Fc protein that prevents IFN gamma from binding to its receptor. Neither approach enhanced TSHR antibody levels, although splenocyte IFN gamma production in response to TSHR antigen was absent (IFN gamma knockouts) or reduced (IFN gamma receptor-Fc). Moreover, production of IL-2, another Th1 cytokine, but not Th2 cytokines, indicated that neither strategy overcame the Th1 bias of im DNA vaccination. Importantly, splenocyte production of IFN gamma and IL-2 provides a sensitive detection system for TSHR-specific T cells. Unexpectedly, higher TSHR antibody levels developed in rare mice. High titer animals had TSHR-specific responses of both Th2 and Th1 types, whereas low titer animals had Th1-restricted TSHR responses. The heterogeneity of responses induced by TSHR DNA vaccination in mice may provide insight into the titers and IgG subclasses of spontaneous autoantibodies in humans.

Animals↗