Substitutions of different regions of the third cytoplasmic loop of the thyrotropin (TSH) receptor have selective effects on constitutive, TSH-, and TSH receptor autoantibody-stimulated phosphoinositide and 3',5'-cyclic adenosine monophosphate signal generation.

Abstract

TSH and immunoglobulin G (IgG) preparations from patients with Graves' disease increase inositol phosphate as well as cAMP formation in Cos-7 cells transfected with rat TSH receptor (TSHR) cDNA. In a previous report, we mutated alanine 623 of the third cytoplasmic loop (residues 605-625) of the TSHR and showed it was critical for TSH and Graves' IgG initiation of phosphatidylinositol bisphosphate (PIP2) but not cAMP signaling. In this report, we substituted residues in the third loop of the TSHR with sequences from the N- and C-termini of the third loop of the alpha 1- and beta 2-adrenergic receptors (ARs), which computer analysis has identified as homologous to those in the TSHR. Alanine 623 is conserved in most ARs as well as in glycoprotein hormone receptors; there is, therefore, no change in alanine 623. After transfection of the mutant TSHR cDNAs into Cos-7 cells, we show that the mutant proteins are normally synthesized, processed, and incorporated into the membrane bilayer by Western blotting with a specific receptor antibody. We also show that the dissociation constant for TSH binding in all mutants is the same or lower than wild type TSHR. We then evaluated the ability of TSH or Graves' IgG to increase PIP2 and cAMP signals in each transfectant. Mutants A622 and B621 replace, respectively, residues 622-625 and 621-625 of the TSHR with alpha 1- and beta 2-AR residues from the C-terminus of the third cytoplasmic loop; mutants A607 and B605 replace, respectively, TSHR residues 607-609 and 605-609 with N-terminus residues from alpha 1- and beta 2-AR. All four mutants, like the alanine 623 mutant, result in transfected cells which lose TSH and Graves' IgG initiation of PIP2 but not cAMP signalling. Like the alanine 623 mutation to glutamic acid, the A607, B605, A622, and B621 mutants also result in decreased basal cAMP, but not inositol phosphate levels, relative to wild type receptor. In contrast to these results, mutants A610, B610, A617, and B617, which replace residues 610-613 or 617-620 of the TSHR with corresponding residues of the alpha 1- and beta 2-AR, retain TSH and Graves' IgG responsiveness in both inositol phosphate and cAMP assays. Mutation of residues 610-613, in fact, potentiates TSH-increased inositol phosphate production, despite having no effect on TSH-increased cAMP production.(ABSTRACT TRUNCATED AT 250 WORDS)

The first cytoplasmic loop of the thyrotropin receptor is important for phosphoinositide signaling but not for agonist‐induced adenylate cyclase activation

“…3B). Thus, WT increased the basal CAMP level compared with the pSG5 control transfectant as previously described [2]. The maximal response compared with the basal levels of the same transfectant in the absence of TSH, was slightly but significantly decreased in B441 (Table 1).…”

“…Localization of sites in TSHR involved in each signal transduction is important for understanding the mechanism of receptor activation. We have investigated the 3rd [2,4] and 2nd [6] cytoplasmic loops of the TSHR by substituting the corresponding sequence from adrenergic receptors (ARs) and have found that: (i) the middle portion of the 2nd loop is important for agonistinduced CAMP production; (ii) widely distributed portions of the both loops are important for PIP* signaling; and (iii) both loops are important for regulating basal CAMP levels.…”

“…Oligonucleotide-mediated, site-directed mutagenesis was used to create the mutants as previously described [2,4,6]. …”

“…All assays were simultaneously initiated 48 h after transfection [2,4,6] and after washing with assay buffer: NaCl-free, Hanks' Balanced Salt Solution containing 0.5% BSA, 222 mM sucrose, and 20 mM HEPES at pH 7.4. ['*'I]TSH binding was measured after incubation for 2 h at 22'C in 1 ml assay buffer containing ['*'I]TSH and 0 to lo-' M unlabeled TSH.…”

Mutations in the Second Cytoplasmic Loop of the Rat Parathyroid Hormone (PTH)/PTH-related Protein Receptor Result in Selective Loss of PTH-stimulated Phospholipase C Activity

“…There are several examples where agonist-stimulated PLC activity of a G proteincoupled receptor is severely affected, when a single amino acid is mutated. The TSH receptor's capacity to generate IPs, for example, was markedly impaired when alanine at position 623 in the 3i loop was mutated to lysine or glutamic acid, but its capacity to activate adenylyl cyclase was unimpaired (48). It is puzzling, however, that the ␣1B-adrenergic receptor became constitutively activated with respect to stimulating PLC, when alanine at position 293, which is located at the same relative position as alanine 623 in the TSH receptor, was replaced with any amino acid (49).…”