Characterization of a library of 23 mutant versions of the ghrelin receptor with substitutions systematically placed throughout the main ligandbinding crevice and in the extracellular part of the receptor
The constructs were expressed in transiently transfected COS-7 cells. In the first column, expression of each mutation is assessed by cell surface ELISA stated as fraction of wild-type receptor expression. In the second column is shown the constitutive activity of the mutant receptors expressed as percentage of basal signaling activity compared to the maximal Ghrelin-stimulated activity The potency (EC50) of the compounds with respect to stimulating inositol phosphate accumulation was determined in cells expressing 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.
B&W | Expression Level | n | Constitutive Activity | n | Ghrelin | n | Fmut | |
|---|---|---|---|---|---|---|---|---|
| % | nM | |||||||
| WT-Ghrelin R1a | 1a | 3 | 46 ± 1 | 52 | 0.34 ± 0.02 | 52 | 1.0 | |
| AspII:20Asn (Asp99) | 2.60 | 0.62 ± 0.15 | 3 | 69 ± 2 | 8 | 1.10 ± 0.2 | 8 | 3.4 |
| PheIII:04Ser(Phe119) | 3.28 | 1.20 ± 0.21 | 3 | 39 ± 2 | 21 | 0.44 ± 0.04 | 17 | 1.3 |
| GlnIII:05Ala(Gln120) | 3.29 | 0.37 ± 0.11 | 3 | 49 ± 3 | 11 | 2.30 ± 0.6 | 11 | 6.8 |
| SerIII:08Ala (Ser123) | 3.32 | 1.20 ± 0.08 | 3 | 36 ± 2 | 16 | 0.77 ± 0.30 | 16 | 2.3 |
| GluIII:09Gln (Glu124) | 3.33 | 0.75 ± 0.18 | 3 | 44 ± 2 | 10 | 52.00 ± 11 | 10 | 150 |
| ThrIII:12Ala (Thr127)b | 3.36 | 0.73 ± 0.17 | 3 | 64 ± 5 | 4 | 0.61 ± 0.22 | 4 | 3.2 |
| SerIV:16Ala (Ser174) | 4.56 | 0.78 ± 0.12 | 3 | 41 ± 3 | 10 | 0.65 ± 0.10 | 10 | 2.1 |
| IleIV:20Ala (Ile178) | 4.60 | 1.00 ± 0.3 | 3 | 46 ± 2 | 10 | 0.82 ± 0.15 | 10 | 2.4 |
| Arg199Leub | 0.94 ± 0.08 | 3 | 31 ± 3 | 7 | 0.76 ± 0.34 | 7 | 2.2 | |
| Glu197Glnb | 0.99 ± 0.04 | 3 | 49 ± 2 | 8 | 2.30 ± 0.9 | 8 | 6.9 | |
| MetV:05Ala (Met213) | 5.39 | 1.30 ± 0.4 | 3 | 49 ± 2 | 13 | 0.44 ± 0.07 | 13 | 1.3 |
| ValV:08Ala (Val216) | 5.42 | 0.53 ± 0.06 | 3 | 53 ± 1 | 11 | 0.91 ± 0.24 | 6 | 2.7 |
| SerV:09Ala (Ser217) | 5.43 | 0.73 ± 0.12 | 3 | 41 ± 2 | 7 | 0.37 ± 0.09 | 7 | 1.1 |
| PheV:12Ala (Phe220) | 5.46 | 0.68 ± 0.07 | 3 | 22 ± 2 | 12 | 0.48 ± 0.08 | 13 | 1.4 |
| PheVI:16Ala (Phe279) | 6.51 | 0.72 ± 0.23 | 3 | 0 ± 1 | 13 | 12.00 ± 3 | 12 | 36 |
| ArgVI20:Gln (Arg283) | 6.55 | 0.67 ± 0.26 | 3 | 20 ± 2 | 12 | 19.00 ± 4 | 10 | 55 |
| PheVI:23Ala (Phe286)b | 6.58 | 1.00 ± 0.20 | 45 ± 2 | 7 | 8.50 ± 2.6 | 7 | 25 | |
| SerVI:24Ala (Ser287)b | 6.59 | 1.10 ± 0.20 | 3 | 50 ± 2 | 7 | 0.57 ± 0.16 | 7 | 1.7 |
| GlnVII:-02Ala (Gln302)b | 7.32 | 0.49 ± 0.06 | 3 | 43 ± 4 | 6 | 1.90 ± 0.7 | 6 | 5.6 |
| AsnVII:02Ala (Asn305) | 7.35 | 1.10 ± 0.2 | 3 | 26 ± 2 | 14 | 1.20 ± 0.3 | 11 | 3.5 |
| PheVII:06Leu (Phe309) | 7.39 | 1.20 ± 0.5 | 3 | 38 ± 3 | 8 | 0.32 ± 0.07 | 8 | 0.9 |
| PheVII:09Ala (Phe312) | 7.42 | 0.64 ± 0.14 | 3 | 24 ± 5 | 8 | 0.88 ± 0.13 | 6 | 2.6 |
| Ξ-35 N-terminal |
| 0.28 ± 0.18 | 3 | 41 ± 4 | 5 | 0.67 ± 0.05 | 5 | 2.0 |