TABLE 2

Characterization of selected EBI2 mutations

Recombinant EBI2 mutant receptors were characterized through receptor expression analyses, [³⁵S]GTPγS assays, and radioligand binding assay. Total expression and cell-surface expression were detected with an anti-V5 antibody in enzyme-linked immunosorbent assays, in the presence or absence of 1% Triton X-100 as the penetration reagent. The expression of N-terminally V5-tagged EBI2 is expressed as 100%. Cells without recombinant protein expression or with C-terminally V5-tagged GPR81 (GPR81-V5) were used as negative controls for protein expression. For the [³⁵S]GTPγS binding assays, EC₅₀ values are the 7α,25-OHC concentrations that stimulated 50% of the maximal response for each individual mutant. E_max values are expressed as percentages of the maximal response of wild-type EBI2. The assays were performed in triplicate, and values represent mean ± S.E.M. For the radioligand binding assays, the total specific binding for each mutant was calculated as a percentage of the maximal specific [³H]7α,25-OHC binding to wild-type EBI2.

Region of Residue Changed	Mutation	Receptor Expression		[³⁵S]GTPγS Binding		Specific Radioligand Binding
Region of Residue Changed	Mutation	Surface	Total	EC₅₀	E_max	Specific Radioligand Binding
		% of WT level		nM	% of WT response	% of WT level
WT EBI2		100.00	100.00	0.11 ± 0.03	100	100.00
N terminus	C21A	20.68	59.97	2.29 ± 0.82	23.36 ± 1.21	5.42 ± 3.39
ECL1	C104A	2.75	90.22	N.A.	N.A.	N.A.
ECL2	C181A	5.49	79.51	N.A.	N.A.	N.A.
ECL2	E183A	59.82	74.26	5.46 ± 2.00	53.90 ± 2.07	11.33 ± 1.47
ECL3	C280A	32.60	53.24	0.93 ± 0.07	50.26 ± 2.00	16.56 ± 3.29
TM2	P85A	17.44	62.42	2.16 ± 1.67	27.28 ± 8.37	6.37 ± 1.43
	Y90A	23.56	80.78	23.74 ± 3.25	39.74 ± 1.81	N.A.
	D77A	86.97	78.50	N.A.	N.A.	99.31 ± 5.86
	R87A	110.84	117.04	49.04 ± 7.72	49.97 ± 1.01	N.A.
	R87W	77.13	144.55	64.01 ± 4.01	76.17 ± 0.86	17.59 ± 8.72
	R87K	75.63	78.64	3.99 ± 0.36	78.21 ± 1.43	78.02 ± 9.35
TM3	N114A	47.63	77.51	N.A.	N.A.	15.98 ± 5.04
	Y112A	91.83	89.34	11.42 ± 2.49	96.01 ± 3.22	9.39 ± 4.04
	Y116A	118.08	110.49	9.13 ± 0.92	51.71 ± 4.99	N.A.
TM5	L197A	105.57	116.52	11.78 ± 0.76	87.93 ± 3.36	20.21 ± 1.77
TM6	F257A	35.86	45.06	0.11 ± 0.21	36.74 ± 3.43	15.85 ± 2.26
	Y260A	114.07	125.93	140.50 ± 6.87	125.90 ± 2.15	N.A.
	H261A	79.95	97.96	63.71 ± 17.19	44.81 ± 3.68	4.65 ± 1.54
	I264A	112.03	104.26	1.72 ± 0.49	73.30 ± 7.87	10.84 ± 3.48
TM7	V294A	74.29	137.79	3.11 ± 4.60	80.37 ± 4.42	47.65 ± 2.24
	M297I	105.34	90.98	4.09 ± 0.42	92.88 ± 2.09	36.95 ± 3.71
	M297A	110.75	142.23	0.68 ± 0.04	158.5 ± 4.63	93.55 ± 5.05

WT, wild-type; N.A., not applicable, because no receptor stimulation by 7α,25-OHC or specific binding was observed.