TABLE 2.
Characterization of a library of 23 mutant versions of the ghrelin receptor with substitutions systematically placed throughout the main ligand-binding crevice and in the extracellular part of the receptor
The constructs were expressed in transiently transfected COS-7 cells. Under the “Ghrelin” column, expression of each mutation as assessed by cell surface ELISA is stated as a fraction of wild-type receptor expression. In the next column, the constitutive activity of the mutant receptors is shown as percent basal signaling activity of the maximal ghrelin-stimulated activity (37). The potency (EC50) of wFw-Isn-NH2 with respect to stimulating inositol phosphate accumulation was determined in cells expressing either the wild-type or mutant form 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.
| Mutation | Ballesteros-Weinstein numbering | Ghrelin |
Constitutive activity |
wFw-Isn-NH2 |
|||||
|---|---|---|---|---|---|---|---|---|---|
| Expression level | n | %a | n | % Emax | EC50 | n | Fmut | ||
| nm | |||||||||
| WT | 1 | 3 | 46 ± 1 | 52 | 100 | 42 ± 5 | 21 | 1,0 | |
| AspII:20Asn | 2.60 | 0,62 ± 0,15 | 3 | 69 ± 2 | 8 | 57 ± 11 | 36 ± 5 | 4 | 0,9 |
| PheIII:04Ser | 3.28 | 1,20 ± 0,21 | 3 | 39 ± 2 | 21 | 77 ± 18 | 28 ± 5 | 4 | 0,7 |
| GlnIII:05Ala | 3.29 | 0,37 ± 0,11 | 3 | 49 ± 3 | 11 | 33 ± 9 | 26 ± 6 | 3 | 0,6 |
| SerIII:08Ala | 3.32 | 1,20 ± 0,08 | 3 | 36 ± 2 | 16 | 47 ± 10 | 100 ± 30 | 4 | 2,4 |
| GluIII:09Gln | 3.33 | 0,75 ± 0,18 | 3 | 44 ± 2 | 10 | 30 ± 2 | 54 ± 17 | 4 | 1,3 |
| ThrIII:12Ala | 3.36 | 0,73 ± 0,17 | 3 | 64 ± 5 | 4 | 110 ± 9 | 8,3 ± 3 | 5 | 0,2 |
| SerIV:16Ala | 4.56 | 0,78 ± 0,12 | 3 | 41 ± 3 | 10 | 72 ± 15 | 120 ± 10 | 3 | 2,9 |
| IleIV:20Ala | 4.60 | 1,00 ± 0,3 | 3 | 46 ± 2 | 10 | 72 ± 18 | 350 ± 13 | 4 | 8,3 |
| R198L | 0,94 ± 0,08 | 3 | 31 ± 3 | 7 | 190 ± 42 | 140 ± 17 | 3 | 3,3 | |
| E196Q | 0,99 ± 0,04 | 3 | 49 ± 2 | 8 | 130 ± 28 | 47 ± 14 | 4 | 1,1 | |
| MetV:05Ala | 5.36 | 1,30 ± 0,4 | 3 | 49 ± 2 | 13 | 81 ± 19 | 74 ± 16 | 4 | 1,8 |
| VaIV:08Ala | 5.42 | 0,53 ± 0,06 | 3 | 53 ± 1 | 11 | 51 ± 15 | 110 ± 10 | 3 | 2,6 |
| SerV:09Ala | 5.43 | 0,73 ± 0,12 | 3 | 41 ± 2 | 7 | 54 ± 12 | 83 ± 15 | 3 | 2,0 |
| PheV:12Ala | 5.46 | 0,68 ± 0,07 | 3 | 22 ± 2 | 12 | 60 ± 19 | 110 ± 20 | 3 | 2,6 |
| PheVI:16Ala | 6.51 | 0,72 ± 0,23 | 3 | 0 ± 1 | 13 | 53 ± 5 | 260 ± 50 | 4 | 6,2 |
| ArgVI20:Gln | 6.55 | 0,67 ± 0,26 | 3 | 20 ± 2 | 12 | 33 ± 15 | 110 ± 80 | 4 | 2,6 |
| SerVI:24Ala | 6.58 | 1,00 ± 0,20 | 3 | 45 ± 2 | 7 | 89 ± 26 | 62 ± 24 | 4 | 1,5 |
| PheVI:23Ala | 6.59 | 1,10 ± 0,20 | 3 | 50 ± 2 | 7 | NA | >1000 | 3 | >24 |
| GlnVII: −02Ala | 7.32 | 0,49 ± 0,06 | 3 | 43 ± 4 | 6 | 92 ± 30 | 62 ± 23 | 4 | 1,5 |
| AsnVII:02Ala | 7.35 | 1,10 ± 0,2 | 3 | 26 ± 2 | 14 | 67 ± 11 | 75 ± 28 | 3 | 1,8 |
| PheVII:06Leu | 7.39 | 1,20 ± 0,5 | 3 | 38 ± 3 | 8 | 87 ± 17 | 1000 ± 200 | 5 | 24 |
| PheVII:09Ala | 7.42 | 0,64 ± 0,14 | 3 | 24 ± 5 | 8 | 17 ± 4 | 91 ± 10 | 3 | 2,2 |
a 100% = maximal ghrelin stimulation of WT receptor.