title: Occupancy of both sites on the thyrotropin (TSH) receptor dimer is necessary for phosphoinositide signaling.
abstract: The thyroid-stimulating hormone (TSH) receptor signals via G(s) to produce cAMP and via G(q/11) to produce inositol-1,4,5-trisphosphate, which is degraded to inositol monophosphate (IP1; phosphoinositide signaling). The potency of TSH for cAMP signaling is higher than for phosphoinositide signaling, and it was suggested that there are "spare receptors" for cAMP signaling. In a human embryonic kidney macrophage scavenger receptor-expressing (HEK-EM) 293 model system, there are no spare receptors, but the cells still exhibited 100-fold differences in potencies. Dose responses for TSH-stimulated dissociation of prebound (125)I-TSH (negative cooperativity; EC(50)=70 mU/ml), which requires TSH binding to both sites of the TSH receptor (TSHR) homodimer, and TSH-stimulated IP1 production (EC(50)=50 mU/ml) were indistinguishable. Fluorescence resonance energy transfer (FRET) using tagged receptors showed that TSHR formed homodimers and heterodimers with two binding-deficient mutant TSHRs, L252P and C41S. When L252P or C41S was expressed with TSHR, that is, when TSHR/L252P or TSHR/C41S heterodimers could only bind one TSH, TSH-stimulated IP1 production was decreased relative to cAMP production. The slopes of linear regression analyses comparing fold stimulation by TSH of IP1 vs. cAMP production were 0.044 ± 0.0047, 0.0043 ± 0.0041, and 0.0059 ± 0.0014 for cells expressing TSHR alone, TSHR and L252P, or TSHR and C41S, respectively. We suggest that TSHR coupling to phosphoinositide signaling is dependent on binding 2 molecules of TSH to TSHR homodimer, causing a conformational change allowing coupling to G(q/11).
authors.affiliation: Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-8029, USA.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Allen
authors.forename: Susanne
authors.initials: S
authors.lastname: Neumann
authors.forename: Marvin C
authors.initials: MC
authors.lastname: Gershengorn
mesh_terms: D000242:Cyclic AMP; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D021122:Protein Subunits; D011989:Receptors, Thyrotropin / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013972:Thyrotropin / Q000378:metabolism
chemical_list: D010716:Phosphatidylinositols; D021122:Protein Subunits; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D000242:Cyclic AMP; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019205:GTP-Binding Protein alpha Subunits, Gs