TABLE 1

¹²⁵I-VIP Binding and adenylyl cyclase assays in CHO cells expressing wild-type and mutated VPAC₁ receptors

Binding parameters (K_D and B_max) of CHO cells expressing wild-type and mutated VPAC₁ receptors were determined by Scatchard analysis. The data is consistent with one binding site for both wild and mutated receptors. VIP stimulation of adenylyl cyclase activity in membranes prepared from transfected CHO cells was also performed. The EC₅₀ values are from VIP stimulation of adenylyl cyclase activity of CHO cell membranes after short-term (5 min) cell exposure to VIP in desensitization studies. The corresponding desensitization curves as well as control curves obtained in the absence of VIP exposure are shown in Fig.2. All data are mean ± S.E. of at least three independent experiments.

			Adenylyl Cyclase Activity
	VIP/VPAC₁ binding				5-Min VIP Treatment
Constructs	K_D	B_max	Basal	Maximal	EC₅₀ Without	EC₅₀ With
	nM	fmol/mg of protein	pmol/min/mg of protein		nM	nM
Wild-type	0.3 ± 0.1	300 ± 22	10.7 ± 0.1	0.27 ± 0.08	0.27 ± 0.08	3.15 ± 0.61^**
IL1/2/3/C-T	0.8 ± 0.1	361 ± 37	9.8 ± 2.1	0.23 ± 0.07	0.23 ± 0.07	0.29 ± 0.07
IL2/3/C-T	0.9 ± 0.2	325 ± 29	10.3 ± 0.2	0.31 ± 0.06	0.31 ± 0.06	0.41 ± 0.11
IL2 (S250A)	0.7 ± 0.1	774 ± 65	9.0 ± 0.3	0.11 ± 0.04	0.11 ± 0.04	1.08 ± 0.21^**
IL3	0.7 ± 0.2	494 ± 36	9.0 ± 0.1	0.20 ± 0.07	0.20 ± 0.07	1.90 ± 0.40^**
C-T (S447A3)	0.5 ± 0.2	500 ± 45	11.8 ± 0.4	0.16 ± 0.05	0.16 ± 0.05	0.16 ± 0.04
C-T (S447A)	0.5 ± 0.2	482 ± 102	10.1 ± 0.2	0.57 ± 0.21	0.57 ± 0.21	0.55 ± 0.16
C-T (S447D)	0.8 ± 0.2	502 ± 39	8.4 ± 1.1	0.37 ± 0.05	0.37 ± 0.05	0.54 ± 0.18

↵** p < 0.01, compared with control EC₅₀ values.