Table 3.
Summary of the polymorphic hMC4R pharmacology of the endogenous C-terminal antagonist agouti-related protein ligand [hAGRP(87-132)] and the synthetic agonist MTII.
| Mutation | TM | MTII Agonist EC50(nM) | Antagonist pA2 hAGRP(87-132) | hAGRP(87-132) Inverse Agonist Activity Observed |
|---|---|---|---|---|
| WT# | 0.011±0.003 | 8.76±0.2 | Yes | |
| R7H | N-Term | 0.017±0.006 | 8.60±0.6 | Yes |
| R18H | N-Term | 0.011±0.007 | 8.79±0.8 | Not observed |
| R18L | N-Term | 0.006±0.002 | 8.88±0.1 | Not observed |
| S36Y | N-Term | 0.011±0.007 | 8.39±0.3 | Yes |
| P48S | 1 | 0.026±0.022 | 8.29±0.7 | Not observed |
| V50M | 1 | 0.015±0.006 | 8.04±0.1 | Yes |
| F51L | 1 | 0.028±0.013 | 8.72±0.2 | Yes |
| E61K | 1 | 0.28±0.0.08* partial agonist | 8.48±0.2 | Not observed |
| I69T | 1 | 0.071±0.001* | 8.35±0.2 | Not observed |
| D90N | 2 | 1.84±0.65* partial agonist | 8.61±0.3 | Not observed |
| S94R | 2 | No stim | ND | ND |
| G98R | 2 | No stim | ND | ND |
| I102T/V103I | 2 | 0.034±0.016 | 8.41±0.3 | Not observed |
| I121T | 3 | 0.010±0.005 | 8.58±0.2 | Not observed |
| A154D | IL2 | 0.038±0.004 | 8.04±0.2 | Not observed |
| Y157S | IL2 | 1.59±0.13* partial agonist | 8.31±0.1 | Not observed |
| W174C | 4 | No stim | ND | ND |
| G181D | 4 | No stim | ND | ND |
| F202L | 5 | 0.010±0.002 | 8.57±0.4 | Yes |
| A219V | 5 | 0.35±0.12* | 7.86±0.1* | Not observed |
| I226T | IL3 | 0.027±0.011 | 8.15±0.1 | Not observed |
| G231S | IL3 | 0.019±0.017 | 8.52±0.4 | Yes |
| G238D | IL3/6 | 0.029±0.013 | 7.74±0.2* | Yes |
| N240S | IL3/6 | 0.010±0.004 | 8.47±0.1 | Not observed |
| 750DELGA | C-Term | No stim | ND | ND |
| C271R | 6/EX3 | 1.62±0.99* partial agonist | 8.87±0.4 | Not observed |
| S295P | 7 | 0.065±0.005* | 7.88±0.3* | Yes |
| P299L | 7 | No stim | ND | ND |
| E308K | C-Term | 0.029±0.004 | 8.86±0.3 | Not observed |
| I317V | C-Term | 0.029±0.022 | 8.67±0.1 | Not observed |
| L325F | C-Term | 0.017±0.013 | 8.47±0.1 | Yes |
Indicates the average from seven independent experiments of HEK-293 cells stably expressing the wild type (WT) hMC4R. The MTII agonist EC50 values indicated represent the mean of at least three independent experiments with the standard error of the mean indicated. No stim indicates that the MTII agonist was unable to stimulate the receptor polymorphisms at up to 1 μM concentrations. Partial agonist indicates that some stimulatory agonist pharmacology resulted, but the maximal stimulation levels were less then the wild type control response. The antagonistic pA2 values were determined using the Schild analysis and the agonist MTII (Ki=-Log pA2). The indicated errors for the functional data (pA2) represent the standard error of the mean determined from at least three independent experiments. ND indicates that the pA2 value could not be determined since MTII was unable to potently stimulated the polymorphic receptor. Statistical analysis was performed using a student T-test compared to the wild type receptor values with
p<0.05.