Table 4.

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

	LOCATION	POINT MUTATION	Univ.Lig Affinity (nM ± SEM)
	Wild type hGRPR 0.1×106	-	0.10 ± 0.01
4	EC2	D97N	1.24 ± 0.01*
80		D97A	1.13 ± 0.06*
81		D97E	0.19 ± 0.02*
10		D104K	0.20 ± 0.01*
82		D104A	0.20 ± 0.01*
83		D104N	0.30 ± 0.02*
14		G112V	2.98 ± 0.15*
84		G112A	0.15 ± 0.01*
16	UTM3	I116A	0.18 ± 0.01*
85		I116K	0.22 ± 0.01*
86		I116L	0.055 ± 0.010
87		I116M	0.051 ± 0.004
18		L121A	0.48 ± 0.02*
88		L121I	0.067 ± 0.012
89		L121K	0.29 ± 0.01*
90		L121M	0.13 ± 0.01
70	EC3	I209W	0.27 ± 0.01*
91		I209A	0.27 ± 0.01*
33		I283A	0.47 ± 0.02*
92	UTM6	I283K	1.75 ± 0.11*
93		I283L	0.22 ± 0.01
94		I283M	0.36 ± 0.01
34		Y284A	5.78 ± 0.33*
95		Y284F	0.16 ± 0.01*
96		Y284L	2.53 ± 0.14*
97		Y284W	4.22 ± 0.26*
35		R287N	7.07 ± 0.47*
98		R287K	1.02 ± 0.05*
75	EC4	D295S	0.18 ± 0.02*
99		D295A	0.46 ± 0.01*
38		H300S	0.99 ± 0.03*
100		H300A	0.15 ± 0.01*
78		V302I	0.35 ± 0.01*
101		V302A	0.060 ± 0.001

Group IV (22 new mutations) include hGRPR point mutants made to explore the important properties of the amino acid replacement made in that position that was found to alter the affinity for [D-Tyr⁶, β-Ala¹¹, Phe¹³, Nle¹⁴]Bn(6-14) [Univ.Lig] and identified in Tables 1–3. Experiments were performed as described in Table 1, and Fig. 1, 3 and 4 Figure legends. The affinities (IC₅₀) were calculated as described in Methods. Each point mutation’s affinity for Univ.Lig was compared to that of cells expressing wild type GRPR. 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.