Báo cáo khoa học: Proper targeting and activity of a nonfunctioning thyroid-stimulating hormone receptor (TSHr) combining an inactivating and activating TSHr mutation in one receptor

Proper Targeting and Activity of a Nonfunctioning Thyroid-Stimulating Hormone Receptor (TSHr) Combining an Inactivating and Activating TSHr Mutation in One Receptor

Tác giả: Patrizia Agretti, Giuseppina De Marco, Paola Collecchi, Luca Chiovato, Paolo Vitti, Aldo Pinchera and Massimo Tonacchera

Lĩnh vực: Endocrinologia e Metabolismo, Ortopedia e Traumatologia, Medicina del Lavoro, Oncologia, Anatomia Patologica, Endocrinologia.

Nội dung tài liệu: Nghiên cứu này tập trung vào việc khám phá cơ chế hoạt động của thụ thể hormone kích thích tuyến giáp (TSHr), đặc biệt là ảnh hưởng của việc kết hợp các đột biến bất hoạt và hoạt hóa trong cùng một thụ thể. Các nhà nghiên cứu đã tạo ra các thụ thể TSHr đột biến kép để đánh giá hoạt tính nội tại (constitutive activity) đối với con đường cAMP và con đường inositol phosphate (IP). Kết quả cho thấy việc kết hợp hai đột biến hoạt hóa làm tăng hoạt tính nội tại của con đường cAMP nhưng không ảnh hưởng đến con đường IP. Điều đáng chú ý là thụ thể kép chứa đột biến bất hoạt T4771 lại thể hiện hoạt tính hoạt hóa với hoạt tính nội tại cho cả hai con đường cAMP và IP. Nghiên cứu cũng chỉ ra rằng một dạng bất hoạt của TSHr có thể đạt được bề mặt màng khi kết hợp với một dạng hoạt hóa của thụ thể.

Mục lục chi tiết:

• Proper targeting and activity of a nonfunctioning thyroid-stimulating hormone receptor (TSHr) combining an inactivating and activating TSHr mutation in one receptor
• Activating mutations of the thyroid-stimulating hormone receptor (TSHr) have been identified as a cause of toxic adenomas. Germline-inactivating TSHr mutations have been described as a cause of congenital hypothyroidism.
• The effects of combining activating and inactivating mutations within a single receptor was studied.
• Constructs were expressed in COS-7 cells and basal and TSH-stimulated cyclic AMP (cAMP) accumulation and inositol phosphate (IP) production were determined.
• The expression at the cell surface was studied both with binding and fluorescence-activated cell scanning analysis.
• Our results show that the effect of combining the two activating mutations is an increase in the constitutive activity only for the cAMP pathway and not for the IP pathway suggesting that different mutations result in receptor conformations with different relative abilities to couple to Gs-alpha or Gq-alpha.
• Surprisingly the double mutant containing the T4771 behaves as an activating receptor with constitutive activity both for the cAMP and IP pathways.
• These data show that an inactive form of the TSHr which is trapped inside a cell after transfection is able to gain the membrane surface when combined with an activated form of the receptor.
• Keywords: TSH receptor; G-protein-coupled receptors; constitutive activity; site-directed mutagenesis; somatic mutations; germline mutations.
• G-protein-coupled, seven transmembrane segment receptors comprise the largest superfamily of proteins in the body [1].
• Many G-protein coupled receptors have a certain basal activity (constitutive activity) and thus can activate G-proteins in the absence of the agonist [1,2].
• Interestingly, it has been encountered that discrete mutations of these receptors are able to dramatically increase this constitutive agonist-independent receptor activity [3].
• The thyroid-stimulating hormone receptor (TSHr), together with the follicle-stimulating hormone (FSH) and the luteinizing hormone (LH) receptors, is a member of a subfamily of seven transmembrane G-protein-coupled receptors, characterized by a large N-terminal extracellular domain involved in hormone binding [4,5]; the receptor is mainly coupled to adenylyl cyclase via G-alpha and, in some species including man, it activates also the inositol phosphate cascade (IP) via a Gq-alpha protein [6–8].
• Current models of G-protein-coupled receptor activation consider that binding of the ligand, within the slit formed by the transmembrane helices (for biogenic amines), and/or to the extracellular loops (for peptide ligands), relieves a built-in negative constraint by stabilizing an active conformation of the receptor [9].
• In this conformation, the new position of the transmembrane helices translates into an increased affinity of the intracellular loops for G-proteins.
• Somatic and germline activating mutations of the TSHr gene have been identified as a major cause of toxic thyroid adenoma [3,10,11] and hereditary or sporadic nonautoimmune toxic thyroid hyperplasia [12,13], respectively.
• On the contrary, inactivating mutations abolishing basal activity or affecting agonist induced response have been described in cases of congenital hypothyroidism with thyroid hypoplasia [14-16].
• All activating TSHr mutations have been shown to activate adenylyl cyclase when expressed in eukaryotic cells [3,10].
• Some of these mutations possess also constitutive activity for the IP pathway [3,11].
• Inactivating mutations of the TSHr gene may be due to truncated forms of the receptor or to point mutations [14-16].
• It has been demonstrated in vitro that point mutations can alter the routing of the receptor to the cell surface, resulting in loss of basal activity and loss of agonist induced cAMP production [14-16].
• In order to study the mechanism of activation of the TSHr we explored the effects of combining previously described mutations within a single receptor.
• We decided to combine two particularly potent activating TSHr mutations and to combine an activating mutation together with an inactivating one.
• Double mutant receptors (containing two activating TSHr mutations) harboring the amino acid substitution P639S (6th transmembrane segment) [17] and I486M (1st extracellular loop) [3], named I486M/P639S, and the double mutant receptor (containing an activating together with an inactivating TSHr mutation) P639S and T4771 (1st extracellular loop) [16], named T4771/P639S, were constructed.
• Constructs were subcloned in the expression vector pSVL and, after transient expression in COS-7 cells, basal and TSH-induced CAMP and IP production were determined.
• Cell-surface expression was evaluated with [125I]bTSH (bovine TSH) binding, an enzyme immunosorbent assay (EIA) and a fluorescence-activated cell scanning (FACS) analysis using different monoclonal antibodies against the TSHr.
• Our results show that the effect of combining the two activating mutations is an increase in the constitutive activity only for the cAMP pathway and not for the IP pathway suggesting that different mutations result in receptor conformations with different relative abilities to couple to Gs-alpha or Gq-alpha.
• Surprisingly the double mutant containing the T4771 behaves as an activating receptor with constitutive activity both for the cAMP and IP pathways.
• These data show that an inactive form of the TSHr which is trapped inside a cell after transfection is able to gain the membrane surface when combined with an activated form of the receptor.
• Materials and methods
• Construction of mutant TSH receptors
• The constructs harboring the single mutated TSH receptors T4771, I486M and P639S have been described [11,16,17].
• In brief, a Spel-CvnI segment (1660-1932) or a CvnI-BstEII segment (1932-2450) in the cDNA of the wild type (WT) TSHr in the expression vector pSVL, was replaced by a homologous segment harboring the mutation in position 477 and 486 or 639, respectively.
• These mutated sequences were cloned directly from DNA extracted from nodular tissues obtained from patients with toxic multinodular goiter (1486M, P639S) or from the blood of a patient with a germline inactivating TSHr mutation (T4771).
• The Spel restriction site in the WT-TSHr was created by site-directed mutagenesis (the change in the coding region does not modify the encoded amino acid sequence).
• For the construction of the double mutants, a CvnI-BstEII fragment in the T477I and I486M TSHr was substituted by homologous segments harboring the P639S mutation, yielding the T477I/P639S or I486M/P639S double mutant, respectively (Fig. 1).
• The resulting constructs were sequenced directly by an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CA, USA) to verify the presence of the mutations.
• Expression in eukaryotic cells of mutated genes
• For transient expression, COS-7 cells were seeded at the concentration of about 150 000 cells per 3-cm dish for binding, EIA and FACS analysis, CAMP and IP determination.
• COS-7 cells were grown in DMEM supplemented with 10% fetal bovine serum, penicillin 100 IU·mL¯¹, streptomycin 100 µg·mL¯¹, fungizone 2.5 µg·mL¯¹ and 1 mm sodium pyruvate.
• One day after seeding, cells were transfected with the DEAE-dextran method followed by a 2-min 10% dimethylsulfoxide shock [18].
• For functional assays, 48 h after transfection cells were used for CAMP or IP determinations and for EIA, FACS analysis and [125I]bTSH binding studies.
• Triplicate dishes were used for each condition and each experiment was repeated at least three times.
• Results were expressed as mean ± SEM from one representative experiment.
• When not shown, SEM values were so small that they fall within the symbols.
• CAMP assay
• Cells were washed with Krebs/Ringer/Hepes buffer (KRH) and preincubated for 30 min at 37 °C.
• This was followed by a 1-h incubation at 37 °C in the presence of 0.5 mm isobutylmethyl xanthine as a cAMP phosphodiesterase inhibitor, in the absence of bTSH (basal values), or in the presence of various concentrations of bTSH (Sigma Chemical Co).
• At the end of the incubation the medium was removed and replaced by 0.1 м HCl.
• The cell extracts were dried in a vacuum concentrator and cAMP was determined as described [17] and expressed as picomoles per dish.
• Inositol phosphate assay
• For IP determinations, 24 h after transfection cells were incubated with 20 µCi·mL¯¹ [3H]inositol (Amersham Pharmacia Biotech Europe, Germany).
• The next day, dishes were washed three times with KRH, preincubated in KRH plus LiCl 10 mm for 30 min at 37 °C and incubated for 18 min at 37 °C in the presence of KRH plus LiCl 10 mм (basal values) or in the same medium plus different concentrations of bTSH.
• The incubation was stopped by addition of ice cold 3% HClO4, and ³H-labeled IP were isolated and assayed by stepwise chromatography on AG1 × 8 resin [19].
• The cell debris in the bottom of the dishes was dissolved in 1 м NaOH and counted as phosphatidyl inositols.
• Results were expressed as percentage radioactivity incorporated in inositol phosphates (IP1 + IP2 + IP3) over the sum of radioactivity in inositol phosphates and phosphatidyl inositols [19].
• Binding assay
• Forty-eight hours after transfection, cells were washed once with Hanks’ solution in which NaCl was replaced by sucrose 280 mm containing 0.2% bovine serum albumin (BSA) and 2.5% low fat milk.
• Binding studies were performed by incubating cells in that same medium at room temperature for 4 h in the presence of about 90 000 c.p.m. [125I]bTSH (a gift of BRAHMS, Berlin, Germany) and the appropriate concentrations of cold bTSH.
• At the end of the incubation cells were rinsed twice with ice cold Hanks’ medium, solubilized with 1 м NaOH and bound radioactivity was determined in a gamma-counter.
• In the absence of a consensus about the bioactivity of pure bovine TSH [20], we have expressed all TSH or TSHr concentrations in mU·mL¯¹, assuming a 1/1 stechiometry for TSH binding to its receptor.
• The competition binding curves have been fitted by nonlinear regression assuming a single receptor site [21].
• Enzyme immunosorbent assay (EIA)
• EIA measurements were carried out with transfected cells, nonpermeabilized and in suspension.
• Forty-eight hours after transfection cells were detached from the plates with NaCl/P; containing 5 mm EDTA and EGTA.
• Cells were then pelleted and incubated with a mouse anti-(human TSHr Ig) (Novocastra Laboratories Ltd, UK) diluted at 1 µg·mL¯¹ in NaCl/P₁ containing 0.5% BSA.
• After two washes with NaCl/P₁, cells were incubated for 1 h at 4 °C with peroxidase-conjugated anti-(mouse IgG) as secondary antibody (Sigma Chemical Co.) diluted 1: 70 000 in NaCl/ P₁ containing 0.5% BSA.
• The cells were washed three times with NaCl/P; and, finally, incubated with the o-phenylene- diamine dihydrochloride substrate (Sigma Chemical Co.) for 30 min at 37 °C.
• The reaction was stopped by adding 1 M H2SO4 and color development was measured at 492 nm.
• FACS analysis
• Cells were detached from culture dishes with 5 mmol·L-¹ each of ethylenediamine tetraacetate and ethyleneglycol- bis-(beta-aminoethyl ether)-N,N,N’,N’-tetraacetic acid in NaCl/P; and transferred to Falcon tubes (2052, Falcon Labware, Cockeysville, MD, USA).
• Cells were washed with NaCl/P; plus 0.1% BSA, centrifuged at 500 g at 4 °C for 3 min, and treated appropriately for the nonpermeabilized or permeabilized cell assay as described previously [16].
• Nonpermeabilized cells were incubated at room temperature for 30 min with 200 µL of a monoclonal antibody directed against the TSHr (BA8 gently gifted from Dr Sabine Costagliola) diluted in NaCl/Pi plus 0.1% BSA.
• A blank sample was prepared by incubating cells with 200 µL NaCl/Pi plus 0.1% BSA.
• For the permeabilized cell assay, cells were fixed with 2% NaCl/Pi/paraformaldehyde (UCB, Brussels, Belgium) and then treated for 30 min with NaCl/P; plus 0.1% BSA and 0.2% saponin (Sigma Chemical Co., St. Louis, MO, USA); all subsequent steps with antibodies were performed in 0.2% saponin.
• Cell-bound monoclonal antibodies were detected washing the cells with NaCl/Pi plus 0.1% BSA and then incubating them for 30 min at 4 °C in the dark with a goat anti-(mouse IgG) fluorescin-conjugated (Becton Dickinson and Co., San Jose, CA, USA) diluted 1 : 20 in NaCl/P₁ with 0.1% BSA containing 10 µg·mL-1 propidium iodide (Sigma).
• Propidium iodide (PI) was used to detect and exclude from the analysis damaged cells.
• Flow cytometric analysis was performed using a FACSort flow cytometer (Becton Dickinson and Co.) equipped with a laser for an excitation at 488 nm to detect monoclonal antibodies conjugated with fluorescin 5-isothiocyanate and PI.
• Fluorescence emission of fluorescin 5-isothiocyanate and PI from single cells were separated and measured using the standard optics of the FACSort.
• The CELLQUEST software program (Becton Dickinson and Co.) was used to acquire and analyze data.
• A minimum of at least 10 000 cells was analyzed.
• Computation of specific constitutive activity (SCA) and relative SCA (RSCA)
• Given that the transfection efficiency for each construct is constant for a given batch of cells, the SCA was calculated by:
• SCA = (Ar – Av)/(Fr – Fv)
• where Ar and Av are the CAMP (or IP) of cells transfected with the mutant constructs and vector, respectively, and Fr and Fv are the corresponding mean fluorescence unit obtained with FACS analysis.
• RSCA, which is normalized to the WT-TSHr, was obtained by:
• RSCA = SCAr/SCA-WT.
• Results
• CAMP cascade
• The effects of each TSH receptor construct harboring one or two mutations have been investigated after transient expression in COS-7 cells.
• For basal CAMP determinations, 250 ng per dish of the DNA of the various constructs, giving maximal cAMP stimulation [3], were used to transfect COS-7 cells.
• As expected, cells transfected with the WT-TSHr exhibited a threefold increase in basal cyclic AMP accumulation with respect to cells transfected with vector alone, showing constitutive activity (Table 1, Fig. 2A).
• Cells transfected with two of the constructs harboring a