Mutational mapping of the binding site for the agonist Ghrelin and the inverse agonists [d-Arg1,d-Phe5, d-Trp7,9,Leu11]substance P (SP-analog) and fQwFwLL using a library of 17 mutant versions of the ghrelin receptor with substitutions systematically placed through the main ligand-binding crevice
The potency (EC50) of the compounds with respect to either stimulating (agonist) or inhibiting (inverse agonist) the constitutive stimulation of inositol phosphate accumulation was determined in COS-7 cells transiently transfected with either the wild-type or the mutant forms of the ghrelin receptor. Fmut indicates the -fold shift in potency induced by the structural change in the receptor compared with the wild-type receptor. In the first column is shown the constitutive activity of the mutant receptors expressed as percentage of basal signaling activity compared with the maximal ghrelin-stimulated activity.
Constitutive Activity | n | Ghrelin | Fmut | SP Analog | Fmut | fQwFwLL | Fmut | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| EC50 | n | EC50 | n | EC50 | n | |||||||||
| % | nM | nM | nM | |||||||||||
| WT-Ghrelin R1a | 42 ± 1 | 53 | 0.34 ± 0.04 | 53 | 18.0 ± 2 | 12 | 21 ± 5 | 15 | ||||||
| AspII:20Asn (Asp99) | 56 ± 5 | 4 | 1.10 ± 0.3 | 4 | 3.2 | 850.0 ± 150 | 3 | 47.00 | 530 ± 230 | 3 | 25.0 | |||
| PheIII:04Ser(Phe119) | 38 ± 4 | 6 | 0.42 ± 0.05 | 6 | 1.3 | 160.0 ± 18 | 5 | 8.90 | 180 ± 24 | 5 | 8.5 | |||
| GlnIII:05Ala(Gln120) | 47 ± 4 | 7 | 2.20 ± 0.8 | 7 | 6.5 | >1000 | >56.00 | >1000 | >47.0 | |||||
| SerIII:08Ala (Ser123) | 35 ± 3 | 8 | 0.32 ± 0.08 | 8 | 0.9 | 4.7 ± 0.2 | 3 | 0.26 | 39 ± 11 | 3 | 1.8 | |||
| GluIII:09Gln (Glu124) | 41 ± 3 | 4 | 86.00 ± 22 | 8 | 250.0 | 260.0 ± 59 | 3 | 14.00 | 170 ± 45 | 3 | 8.0 | |||
| SerIV:16Ala (Ser174) | 43 ± 2 | 8 | 0.47 ± 0.07 | 8 | 1.4 | 1.0 ± 0.3 | 4 | 0.06 | 68 ± 12 | 3 | 3.2 | |||
| IleIV:20Ala (Ile178) | 46 ± 2 | 11 | 0.72 ± 0.11 | 11 | 2.5 | 400.0 ± 40 | 4 | 22.00 | >1000 | 3 | >47.0 | |||
| MetV:05Ala (Met213) | 43 ± 6 | 8 | 0.40 ± 0.04 | 8 | 1.2 | 8.4 ± 1.3 | 3 | 0.47 | 64 ± 4 | 3 | 3.0 | |||
| ValV:08Ala (Val216) | 52 ± 2 | 5 | 1.00 ± 0.3 | 5 | 2.9 | 2.7 ± 0.8 | 3 | 0.15 | 49 ± 1 | 3 | 2.3 | |||
| SerV:09Ala (Ser217) | 46 ± 4 | 4 | 0.39 ± 0.13 | 4 | 1.1 | 11.0 ± 3 | 3 | 0.61 | 98 ± 11 | 4.7 | ||||
| PheV:12Ala (Phe220) | 20 ± 1 | 12 | 0.44 ± 0.07 | 12 | 1.3 | 1.9 ± 0.3 | 4 | 0.11 | 14 ± 4 | 4 | 0.67 | |||
| TrpVI:13Alaa (Trp276) | 4 | 3.2 ± 0.8 | 3 | 9.4 | (13)a | (5.0)a | ||||||||
| PheVI:16Ala (Phe279) | 2 ± 2 | 10 | 14.00 ± 3 | 10 | 41.0 | |||||||||
| ArgVI20:Gln (Arg283) | 17 ± 4 | 7 | 13.00 ± 2 | 7 | 38.0 | 74.0 ± 2 | 3 | 4.10 | 160 ± 50 | 4 | 7.6 | |||
| AsnVII:02Ala (Asn305) | 16 ± 2 | 5 | 2.90 ± 0.5 | 5 | 8.5 | 14.0 ± 6 | 3 | 0.78 | 110 ± 30 | 3 | 5.2 | |||
| PheVII:06Leu (Phe309) | 42 ± 2 | 6 | 0.50 ± 0.12 | 6 | 1.5 | 120.0 ± 32 | 5 | 6.70 | >1000 | 3 | >47.0 | |||
| PheVII:09Ala (Phe312) | 15 ± 1 | 8 | 1.30 ± 0.2 | 8 | 3.8 | 46.0 ± 7 | 3 | 2.60 | >1000 |
| >47.0 | |||
↵ a Because of the lack of constitutive activity of TrpVI:13Ala, the inverse agonists are tested in competition binding using [35S]MK-677 as a radioligand.