TABLE 2

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.