TABLE 2

Mutational mapping of the binding site for the ghrelin receptor agonists, GHRP-6, L-692,429, MK-677, SM-157740, and SM-130686, using a library of 23 mutant versions of the ghrelin receptor with substitutions systematically placed through the main ligand-binding crevice and in the extracellular part of the receptor

The potency (EC50) of the compounds with respect to stimulating 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 as compared to the wild-type receptor. In the first column is shown the Fmut value of ghrelin in the respective ghrelin receptor mutation.



B&W

Ghrelin Fmut

GHRP-6

L-692,429

MK-677

SM-157740

SM-130686

EC50
n
Fmut
EC50
n
Fmut
EC50
n
Fmut
EC50
n
Fmut
EC50
n
Fmut
nM nM nM nM nM
WT-Ghrelin R1a 1.0 1.8 ± 0.21 18 1.0 39.0 ± 7 11 1.0 0.30 ± 0.03 21 1.0 0.49 ± 0.08 18 1.0 0.31 ± 0.04 20 1.0
AspII:20Asn (Asp99) 2.60 3.4 11.0 ± 2.0 3 5.9 6.3 ± 1.7 4 0.2 400.00 ± 300 3 1300.0 9.80 ± 0.7 3 20.0 130.0 ± 90 4 410.0
PheIII:04Ser(Phe119) 3.28 1.3 2.7 ± 0.6 3 1.5 24.0 ± 8 3 0.6 1.30 ± 0.2 5 4.4 2.00 ± 0.7 3 4.1 2.50 ± 0.5 3 8.2
GlnIII:05Ala(Gln120) 3.29 6.8 51.0 ± 2.0 3 28.0 91.0 ± 21 4 2.3 3.60 ± 1.5 4 12.0 2.10 ± 0.7 3 4.2 2.00 ± 0.8 3 6.5
SerIII:08Ala (Ser123) 3.32 2.3 1.5 ± 0.4 6 0.8 89.0 ± 26 4 2.3 2.90 ± 1.1 6 9.7 0.57 ± 0.14 4 1.2 0.87 ± 0.20 3 2.8
GluIII:09Gln (Glu124) 3.33 150.0 >10.000a 4 >5400.0 >10.000a 4 >250.0 >1000a 4 >3300.0 140.00 ± 40 5 290.0 320.0 ± 90 4 1100.0
ThrIII:12Ala (Thr127) 3.36 3.2 6.0 ± 3.0 3 3.2 930.0 ± 250 4 23.0 1.20 ± 0.2 3 4.0 0.53 ± 0.26 3 1.1 1.10 ± 0.1 3 3.6
SerIV:16Ala (Ser174) 4.56 2.1 3.9 ± 2.7 4 2.1 100.0 ± 30 6 2.7 0.38 ± 0.13 4 1.3 0.61 ± 0.10 3 1.3 1.30 ± 0.1 3 4.3
IleIV:20Ala (Ile178) 4.60 2.4 230.0 ± 20.0 3 130.0 >1000a 4 >25.0 1.60 ± 0.6 4 5.3 4.40 ± 1.2 3 8.9 23.00 ± 15 3 76.0
Arg199Leu 2.2 5.6 ± 0.8 4 3.0 180.0 ± 60 3 4.6 0.22 ± 0.02 3 0.7 0.34 ± 0.15 3 0.7 0.36 ± 0.01 3 1.1
Glu197Gln 6.9 2.7 ± 0.9 3 1.5 43.0 ± 7 3 1.1 1.30 ± 0.8 3 4.4 0.57 ± 0.15 3 1.1 0.55 ± 0.20 3 1.8
MetV:05Ala (Met213) 5.39 1.3 4.1 ± 1.0 6 2.2 77.0 ± 18 5 2.0 0.18 ± 0.05 3 0.6 0.89 ± 0.22 3 1.8 0.64 ± 0.21 3 2.4
ValV:08Ala (Val216) 5.42 2.7 5.3 ± 2.3 3 2.9 130.0 ± 4 3 3.2 0.67 ± 0.17 4 2.2 0.48 ± 0.20 3 1.0 0.38 ± 0.19 3 1.2
SerV:09Ala (Ser217) 5.43 1.1 4.4 ± 1.2 3 2.4 38.0 ± 10 3 1.0 0.30 ± 0.07 4 1.0 0.55 ± 0.12 3 1.1 1.60 ± 0.4 3 5.2
PheV:12Ala (Phe220) 5.46 1.4 7.8 ± 1.4 3 4.2 310.0 ± 170 3 8.0 0.27 ± 0.10 6 0.9 0.68 ± 0.13 3 1.4 0.33 ± 0.05 3 1.1
PheVI:16Ala (Phe279) 6.51 36.0 >10,000a 3 >5400.0 >10,000a 4 >250.0 36.00 ± 6 3 120.0 13.00 ± 2 3 27.0 58.00 ± 12 3 190.0
ArgVI20:Gln (Arg283) 6.55 55.0 >10,000a 3 >540.0 >10,000a 4 >250.0 57.00 ± 23 3 190.0 110.00 ± 20 3 230.0 340.00 ± 90 3 1100.0
PheVI:23Ala (Phe286) 6.58 25.0 27.0 ± 7.0 4 14.0 >1000a 4 >25.0 4.30 ± 0.1 3 14.0 6.50 ± 4.2 3 13.0 59.50 ± 21 3 190.0
SerVI:24Ala (Ser287) 6.59 1.7 3.1 ± 1.0 4 1.7 22.0 ± 9 3 0.6 0.60 ± 0.21 4 2.0 1.30 ± 0.5 3 2.5 1.70 ± 0.9 3 5.2
GlnVII:-02Ala (Gln302) 7.32 5.6 8.9 ± 1.1 3 4.8 45.0 ± 18 3 1.1 0.95 ± 0.22 3 3.2 0.94 ± 0.22 3 1.9 0.72 ± 0.14 3 2.3
AsnVII:02Ala (Asn305) 7.35 3.5 125.0 ± 10 6 68.0 >10.000a 4 >250.0 3.50 ± 1.1 3 12.0 0.67 ± 0.10 4 1.4 0.69 ± 0.35 3 2.2
PheVII:06Leu (Phe309) 7.39 0.9 28.0 ± 14.0 5 15.0 260.0 ± 50 3 6.7 0.40 ± 0.08 5 1.3 1.10 ± 0.2 3 2.2 1.00 ± 0.3 3 3.3
PheVII:09Ala (Phe312) 7.42 2.6 32.0 ± 10.0 3 18.0 85.0 ± 16 3 2.2 1.20 ± 0.3 3 4.1 >1000a 3 >2000.0 >1000a 4 >3200.0
Ξ-35 N-terminal

2.0
3.0 ± 0.9
3
1.6
22.0 ± 7
3
0.6
0.36 ± 0.05
4
1.2
0.69 ± 0.12
3
1.4
0.52 ± 0.16
4
1.7
  • B&W, the nomenclature as described by Ballesteros and Weinstein

  • a The EC50 values have been estimated based on graphic illustration of the dose-response curve. EC50 > 10,000 nM means that only the highest concentration applied (10−5 M) increases slightly above basal.