Graves' immunoglobulins activate phospholipase A(2) by recognizing specific epitopes on thyrotropin receptor

Thyroid-stimulating IgG from Graves' patients bind to the TSH receptor and activate both adenylyl cyclase (AC) and phospholipase A(2)(PLA(2)) in FRTL5 thyroid cells. Both activities have been associated with increased thyroid cell growth and function; evidence exists that subpopulations of Graves' I gG can stimulate either AC or PLA(2) cascades and that the activation of bo th is associated with the largest goiters in patients. Studies using chimer as of the human TSHR receptor (hTSHR) and the LH-CG receptor show that most patients with Graves' disease have cAMP-stimulating IgG that require epito pes on the N-terminal portion of the TSHR extracellular domain; epitopes as sociated with PLA(2) activation are not clear. To address this question we used stably transfected Chinese hamster ovary (CHO) cells containing the wi ld-type hTSHR and the hTSHR chimera with residues 8-165 (Mc1+2) substituted by equivalent residues of the LH-CG receptor. PLA, activity, measured as a rachidonic acid (AA) release, was determined in 32 patients with Graves' di sease. We show that 72% of Graves' patients have IgG able to stimulate PLA, in CHO cells transfected with the TSHR and that AA release induced by Grav es' IgG was significantly reduced (P = 0.022) in the CHO-Mc1+2- transfected cells (193 +/- 88% vs. 131 +/- 67%, respectively). Unlike IgG, the effect of TSH was not modified in the CHO-Mc1+2-transfected cells. When we compare d the AC- and PLA,-stimulating activities of these 32 IgG in wild-type TSHR transfectants, we found that 63% of Graves' patients have antibodies able to stimulate both PLA, and AC, whereas some patients' IgG were active only in AC or PLA, assays. Of the patients with IgG having activity in both assa ys in wild-type TSHR transfectants, 50% of the IgG lost their stimulatory a ctivities in both AA release and cAMP assays in Mc1+2 cells. Of the remaind er, some IgG maintained their activity in one (AA release) or the other (cA MP) assay when measured in Mc1+2 chimeras. Thus, our data show that the N-t erminal portion of extracellular domain of the TSHR is required for PLA, as well as AC activation by IgG from patients with Graves' disease. These dat a also demonstrate that patients with Graves' disease have heterogeneous au toantibodies that selectively activate AC and PLA, pathways and suggest tha t patients with autoantibodies active in both assays have more severe disea se, with higher thyroid hormone levels and larger goiters.