Table 1.

Affinities of hGRPR point mutants in Group I for [D-Tyr⁶, β-Ala¹¹, Phe¹³, Nle¹⁴]Bn(6-14) [Univ.Lig]

No.	LOCATION	POINT MUTATION	Univ.Lig Affinity (nM ± SEM)
	Wild type hGRPR 0.1×106	-	0.100 ± 0.006
1	UTM2	L90A	0.053 ± 0.003
2		T92A	0.53 ± 0.03*
3		V96A	0.13 ± 0.42
4	EC2	D97N	1.24 ± 0.01*
5		A98L	0.031 ± 0.007
6		S99I	0.010 ± 0.013
7		R100A	0.094 ± 0.004
8		Y101N	0.038 ± 0.001
9		Y101A	0.120 ± 0.01
10		D104K	0.20 ± 0.01*
11		R110S	0.074 ± 0.009
12		R110A	0.087 ± 0.003
13		R110D	0.026 ± 0.001
14		G112V	2.98 ± 0.15*
15	UTM3	L115A	0.050 ± 0.031
16		I116A	0.18 ± 0.01*
17		I119A	0.11 ± 0.01
18		L121A	0.48 ± 0.02*
19	UTM4	A172T	0.078 ± 0.009
20		I173T	0.066 ± 0.008
21		E175Q	0.15 ± 0.01*
22		A176G	0.12 ± 0.01
23	EC3	F193A	0.078 ± 0.011
24		Y199L	0.069 ± 0.003
25		P200L	0.064 ± 0.004
26		S202N	0.056 ± 0.003
27		L205A	0.096 ± 0.004
28		M212F	0.10 ± 0.01
29	UTM5	F215A	0.063 ± 0.005
30		F218A	0.074 ± 0.001
31	UTM6	N280A	0.037 ± 0.003
32		H281A	0.22 ± 0.01*
33		I283A	0.47 ± 0.02*
34		Y284A	5.78 ± 0.33*
35	EC4	R287N	7.07 ± 0.47*
36		Y289A	0.063 ± 0.008
37		M298P	0.22 ± 0.01*
38		H300S	0.99 ± 0.03*
39		S304A	0.10 ± 0.01
40	UTM7	I305A	0.14 ± 0.01
41		R308A	0.28 ± 0.02*

Group I (n=41) comprise point mutants made in a hGRPR amino acids which are present in all BnR’s with high affinity for [D-Tyr⁶, β-Ala¹¹, Phe¹³, Nle¹⁴]Bn(6-14) [Univ.Lig] but which different from that in a similar position in hCCK_AR identified as described in Methods. The affinities (IC₅₀) were measured in whole cell radioligand binding assays using 50 pM ¹²⁵I- [D-Tyr⁶, β-Ala¹¹, Phe¹³, Nle¹⁴]Bn(6-14) [Univ.Lig] as described in Methods and Fig. 3 legend. Each point mutation’s affinity for Univ.Lig was compared to that of cells expressing wild type GRPR which bound comparable amounts of radiolabeled ligand. Data are partially from the dose-inhibition curves of representative mutants shown in Fig. 3. IC₅₀’s, the concentration causing half-maximal inhibition, were calculated from the dose-inhibition curves of [D-Tyr⁶, β-Ala¹¹, Phe¹³, Nle¹⁴]Bn(6-14) [Univ.Lig] as described in Methods. Values are means ± SEM from three experiments for those mutants showing no affinity change and 4 to 6 experiments for any showing differences from control and in each experiment each point was measured in duplicate.

^*(in bold)=p<0.05 compared to wild type hGRPR expressed at similar expression levels.