Enhanced cAMP Accumulation by the Human Thyrotropin Receptor Variant with the Pro52Thr Substitution in the Extracellular Domain

Abstract

Recently, a naturally occurring variant of the human thyrotropin receptor with a Pro52Thr substitution in the N-terminal extracellular domain of the receptor has been identified. To determine the functional significance of this substitution, cDNAs of wild-type and variant thyrotropin receptors were stably expressed in Chinese hamster ovary cells. The Pro52Thr substitution did not affect synthesis and membrane localization of the receptor, as evidenced by '251-thyrotropin binding analysis to intact cells. The variant receptor and the wild-type receptor were expressed in equivalent numbers and displayed identical binding affinity for thyrotropin. Strikingly, thyrotropin increased cAMP accumulation to a much greater extent in cells expressing the variant receptor as compared to the wild-type receptor-expressing cells. Basal and cholera toxin-stimulated or forskolin-stimulated cAMP levels were not different. It is concluded that the Pro52Thr substitution in the N-terminal region of the human thyrotropin receptor produces a receptor protein with enhanced coupling to cAMP production. This naturally occurring hyperactive thyrotropin receptor may participate in hyperthyroidism of patients with Graves' disease.Keywords: thyrotropin receptor variant; CAMP; Chinese hamster ovary cells ; Graves' disease.Thyrotropin exerts its effects on the thyroid cell by binding to specific receptors on the cell surface. The thyrotropin receptor belongs to the superfamily of seven-transmembrane receptors that activate signal-transduction pathways through their interaction with guanine-nucleotide-binding regulatory proteins (G proteins) [I -41. The receptor represents one member of a subfamily of glycoprotein hormone receptors that also includes the follitropin receptor and the lutropin receptor [5-lo], and members of this subfamily are characterized by a large N-terminal extracellular domain. As demonstrated for the thyrotropin receptor, this extracellular domain is essential for ligand binding and has also been implicated in signal transduction [I1 -1 31. In contrast to the glycoprotein hormone receptors, the majority of G-protein-coupled receptors, such as rhodopsin, adrenergic, and muscarinic receptors, have a short N-terminal region that does not play a major role in ligand binding and signal transduction [14, 151. In both subgroups, however, the cytoplasmic regions play a pivotal role in G protein coupling and subsequent effector activation [14-181. The mechanism by which binding of thyrotropin to the extracellular region of the receptor is transduced via the membrane-spanning regions to the cytoplasmic segments of the receptor that interact with the G proteins is unknown. In human thyroid cells, signal transduction by the thyrotropin receptor is predominantly mediated by the stimulatory G, protein resulting in enhanced cAMP synthesis by adenylyl cyclase, hormone secretion, and proliferation [ 191.The thyrotropin receptor plays a pivotal role in function and growth of thyroid cells [19]. The receptor is also thought to be the central...

Prevalence of mutations in TSHR, GNAS, PRKAR1A and RAS genes in a large series of toxic thyroid adenomas from Galicia, an iodine-deficient area in NW Spain

“…D36H and P52T, in the extracellular portion of the TSHR, have been previously reported in European patients with Graves' disease and in healthy controls (25). Neither D36H nor P52T have been shown to have any differences either in TSHR binding (26,27) or basal cAMP production level (28), compared with the TSHR wild-type. The third polymorphism that we found, D727E, showed a frequency close to the one reported in German patients (28).…”

Functional Characteristics of a Variant Thyrotropin Receptor

“…Together with the variability of resistance to geneticin of individual transfectants, this constitutes a selection pressure which gives no guarantee that clones picked up individually will behave in an identical way. The use of pools of transfectants is sometimes used to obviate this problem (Chazenbalk et al, 1990) but this was not so in the experiment performed by Loos et al (1995). Despite some other drawbacks (e.g.…”

“…Polymorphic variants of the thyrotropin receptor have been described: a first germline variant, in which residue 36 is replaced by His, was found in patients with Graves' disease but also in healthy people (Heldin et al, 1991 ;Gustavsson et al, 1995); the second variant described, in which Pro52 was replaced by Thr ([Thr52]thyrotropin receptor) was found in patients with Graves' disease but also in 12% of the normal population (Bohr et al, 1993;Baan et al, 1994;Sunthornthepvarakul et al, 1994;Watson et al, 1995). A recent study by Loos et al (1995) Ahhreviufions. G protein, heterotrimeric regulatory guanine-nucleotide-binding protein; Gs, a G-protein subtype known to be involved in stimulation of adenylyl cyclase: Gq, a G-protein subtype demonstrated to allow receptor coupling to phospholipases of the b subclass in a pertussin-toxin-insensitive manner; CHO, Chinese hamster ovary ; COS-7, simian-virus-40-transformed African green monkey kidney.…”

Genetic variation in thyroid hormone pathway genes; polymorphisms in the TSH receptor and the iodothyronine deiodinases

“…The TSHR-Asp727Glu polymorphism is found to be within a linkage disequilibrium block starting at intron 8 and extending about 10 kb beyond the 3 0 UTR of the TSHR gene (22). Conflicting data are also available regarding the response of the TSHR-Pro52Thr variant to TSH stimulation (23)(24)(25). This might reflect the subtle effects of these polymorphisms.…”