1 SUPPLEMENTARY FIGURES Supplementary Figure S1 Supplementary Figure S1. Constitutively active receptors decreased negative cooperativity is not related to changes in dimerization or receptor trafficking. 0.2 nM [ 125 I]-bTSH spontaneous dissociation was measured after 4 hours incubation with the tracer in whole COS-7 cells (a) or membrane (c and inset) enriched fractions [prepared as described here (Perret et al., 1990)] by dilution in reaction buffer and total binding (Bt) over initial binding (Bo) was plotted over time for wt or constitutively active TSHr transiently expressing. TSH induced tracer dissociation is shown as reference. (b) HTRF performed in a subset of constitutively active or wt TSHr transiently expressing HEK 293T cells by incubation of anti-RT labelled with either the donor (europium cryptate) or the acceptor (AF647) for 1 hour at 37 ºC. (c inset): Correlation between basal cAMP accumulation (SCA) and the extent of negative cooperativity (percentage of dissociation at 120 min) of wt (black dot) or constitutively active TSHr expressing COS-7 membrane enriched fraction. Spearman’s coefficient: -0.86, P value= 0.0012. Representative experiments are shown out of at least two independent experiments performed with duplicate or triplicate samples. Data are presented as Mean ± SEM and exponential decay (a, c and inset) non linear regression or linear regression (b) was used to fit the corresponding experimental observation.
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SUPPLEMENTARY FIGURES Supplementary Figure S1
Supplementary Figure S1. Constitutively active receptors decreased negative cooperativity is not related to changes in dimerization or receptor trafficking. 0.2 nM [125I]-bTSH spontaneous dissociation was measured after 4 hours incubation with the tracer in whole COS-7 cells (a) or membrane (c and inset) enriched fractions [prepared as described here (Perret et al., 1990)] by dilution in reaction buffer and total binding (Bt) over initial binding (Bo) was plotted over time for wt or constitutively active TSHr transiently expressing. TSH induced tracer dissociation is shown as reference. (b) HTRF performed in a subset of constitutively active or wt TSHr transiently expressing HEK 293T cells by incubation of anti-RT labelled with either the donor (europium cryptate) or the acceptor (AF647) for 1 hour at 37 ºC. (c inset): Correlation between basal cAMP accumulation (SCA) and the extent of negative cooperativity (percentage of dissociation at 120 min) of wt (black dot) or constitutively active TSHr expressing COS-7 membrane enriched fraction. Spearman’s coefficient: -0.86, P value= 0.0012. Representative experiments are shown out of at least two independent experiments performed with duplicate or triplicate samples. Data are presented as Mean ± SEM and exponential decay (a, c and inset) non linear regression or linear regression (b) was used to fit the corresponding experimental observation.
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Supplementary Figure S2
Supplementary Figure S2. Decreased negative cooperativity of mutant receptors even when expressed at levels similar to the wt. Constitutively active receptors with wt surface expression have increased initial binding. 0.2 nM [125I]-bTSH spontaneous dissociation was measured after 4 hours incubation with the tracer in COS-7 cells transiently expressing wt or constitutively active TSHr by dilution in reaction buffer or a large excess of unlabeled TSH and total binding (Bt) over initial binding (Bo) was plotted over time for receptor constructs transfected with the aim to achieve similar Bo (a-b) or surface expression (c-d). Surface wt or mutant receptor expression was evaluated in COS-7 transfected in parallel by FACS with anti-TSHr monoclonal BA8 as primary antibody and an anti-mouse-phycoerythrin as a secondary probe. (Insets b and d): Correlation between basal cAMP accumulation (SCA) and the extent of negative cooperativity (percentage of dissociation at 120 min) of wt (black dot) or constitutively active TSHr expressing COS-7 cells (color coded). Spearman coefficients -0.91 and P value= 0.0003 for similar Bo correlation (inset b) and -0.86 and P value= 0.0012 for the normalized surface expression correlation (inset d).
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Supplementary Figure S3.
Supplementary Figure S3. Different constitutive activity across wt human GpHrs. Basal cAMP accumulation and surface expression were quantified as explained in Material and Methods for wt TSHr (red), LH/CGr (blue) and FSHr (green) and constitutive cAMP was plotted over surface expression.
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Supplementary Note 1. Tracer binding to a free homomeric receptor Symbols R: homomeric receptor L: unlabeled ligand L*: radioligand Parameters kon1 and koff1: association and dissociation kinetic constants describing the binding of the ligand to one site within the homomer when the other site is free. The corresponding equilibrium dissociation constant is: Kd1 = koff1/kon1 Model Because of the low concentration of radioligand used in the preincubation, it is considered that the two sites of the homomer cannot be simultaneously occupied by the radioligand. The possibility is not considered that binding of a single G protein per homomer could introduce a long lasting asymmetry in the complex. Therefore:
Equations The kinetics obey the following differential equation: d(L*R)/dt = kon1(L*)(R) - koff1(L*R) The bound radioligand concentration (B) is equal to (L*R). At time zero, the experimental constraint is (L*R)0=0. The solution is an exponential function of time with a observed kinetic constant kobs equal to kon1(L*)+koff1. Computer fitting of the radioligand association time course leads to the estimate of kobs. Data are presented in Figure 2F and Table I. Since koff1 is estimated in the chase experiment (see below), kon1 is given by (kobs- koff1)/L*. Supplementary Note 2. Tracer dissociation in the presence of increasing concentrations of unlabeled ligand Parameters kon1 and koff1: association and dissociation kinetic constants describing the binding of the ligand to one site within the homomer when the other site is free. kon2 and koff2: association and dissociation kinetic constants describing the binding of the ligand to one site of the homomer when the other site is already occupied. The corresponding equilibrium dissociation constants are: Kd1 = koff1/kon1 and Kd2 = koff2/kon2 Model As stated before, it is considered that the low radioligand concentration used is unable to bind simultaneously to both sites of the homomer. The time course of the radioligand dissociation induced by the addition of the unlabelled ligand can be analysed on the basis of the following minimal model:
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Equations The kinetics obeys a system of two differential equations: d(L*R)/dt = - koff1(L*R)-kon2(L*R)(L)+koff2(L*RL) d(L*RL)/dt = kon2(L*R)(L)-2koff2(L*RL) The bound radioligand concentration (B) is equal to (L*R) + (L*RL). At time zero, the experimental contraints are (L*R)0=B0 (i.e. the value obtained at the end of the preincubation) and (L*RL)0=0. The solution is a sum of two exponential functions. Thus, either the residual radioligand binding or the rate of radioligand dissociation (see Material and Methods) are described by a two component exponential function of time. The fastest component essentially reflects the association of the unlabelled ligand added at time zero. The slowest component is characterized by the so-called "observed kinetic constant" kobs which depends on the unlabelled ligand concentration (L) used in the experiment. Thus, for each L concentration, the kobs estimate was obtained by fitting the experimental time courses by a sum of two exponential functions. The different kobs estimates were then plotted against L (see Figure 2). The analytical standard resolution of the system of two differential equations leads to the relationship between kobs (reciprocal of time constant) and the kinetic parameter constants:
where β = koff1 + kon2(L)+2koff2 and γ = 2(koff2)2 + koff2kon2(L).
This function is then used to fit the experimental data kobs versus L (Figure 2) in order to estimate koff1, kon2 and koff2 (see Table I).
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SUPPLEMENTARY REFERENCE 61. Perret J, Ludgate M, Libert F, Gerard C, Dumont JE, Gilbert V, and Parmentier
M (1990) Stable expression of the human TSH receptor in CHO cells and characterization of differentially expressing clones. Biochemical and Biophysical Research Communications, 171, 1044-1050.
