Table I. Identification of eight residues of TSHr conferring individually a gain of sensitivity towards hCG.

Code	Description	LRR	FACS		hCG screening		Emax hCG		EC50 hCG
			% expression of wt		100 IU/ml		1000 IU/ml or plateau		IU/ml
			Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
LH/CGr wt			100						0.051	0.006
TSHr wt			100		1.14	0.03	1.30	0.04	ND
Mutants positive in screening
X5 mutants
T8	E61Y	1	75	6	3.50	0.42	3.96	0.52	ND
T9	I85Q	2	107	8	1.53	0.09	2.06	0.11	ND
T11	K209E	7	91	10	4.09	0.27	4.60	0.29	25	4
X2-X3-X4 mutants
T14	T56R+K58S+I60A	1	38	6a	2.38	0.24	2,55	0.04a	88	12a
T15	R80K+Y82E	2	110	18	2.53	0.38	3.00	0.53a	ND
T19	A204S+Y206E+N208K	7	28	7	8.04	2.09	6.12	0.66	31	6
Residues implicated in the gain of sensitivity towards hCG of the X2-X3-X4 mutants
T26	K58S	1	95	2	2.08	0.14	2.72	0.32	43	6
T28	R80K	2	100	5a	1.59	0.03a	1.72	0.06	ND
T29	Y82E	2	87	14a	1.40	0.36a	2.20	0.11	ND
T34	Y206E	7	67	3	2.91	0.29	4.18	0.41	ND
T35	N208K	7	46	3	2.44	0.20	4.07	0.46	ND

List and characteristics of the mutants displaying a gain of sensitivity to hCG. Column 1: code name of the mutants. Column 2: full description of the mutants, with numbering as in Figure 2. Column 3: indication of the LRR β-strands bearing the mutations. Column 4: amount of mutant receptors present at the surface of transfected COS-7 cells (in % of wild type, see Materials and methods). Column 5: fold stimulation of cAMP accumulation over basal, in transfected COS cells after stimulation by 100 IU/ml of hCG. Column 6: fold stimulation of cAMP accumulation over basal, but for stimulation by 1000 IU/ml of hCG, or by a concentration giving a maximal (plateau) response of cAMP accumulation. Column 7: EC₅₀ values in IU/ml for stimulation by hCG of mutant TSHr sensitive enough to obtain a plateau.

^aTwo measurements were performed (results expressed as mean ± range of duplicates). ND, EC₅₀ not determined because concentration–action curve does not reach a plateau of maximal stimulation.