Table 1.
Functional characteristics of Myc-tagged MC2R and mutants coexpressed with MRAPb
| Myc-MC2R | T1/2 cAMP (min) | Rmax cAMP (% of WT)& | R60, cAMP (% of initial rate) | Normalized surface expression with MRAPβ (% of WT) | R5, surface (% of initial rate) | RSR5 (% of initial) | RSR60 (% of WT)& |
|---|---|---|---|---|---|---|---|
| WT | 12.5 ± 1.4# | 100 | 3.19 ± 1.04# | 100 | 33.1 ± 5.4# | 85 ± 2# | 78 ± 1# |
| T131A | 13.2 (10.9, 16.9) | 91 ± 4 | 4.31 ± 0.04 | 127 ± 14a | <0.1c | 92 ± 4 | 95 ± 2c |
| T131D | 4.5 (3.2, 7.7)a | 22 ± 1c | <0.01c | 130 ± 14b | <0.1c | 97 ± 3c | ∼100c |
| S140A | 14.8 (13.2, 17) | 94 ± 3 | 6.05 ± 0.06 | 91 ± 8 | 25.5 ± 0.1 | 78 ± 1 | 72 ± 1c |
| S140D | 11.5 (9.5, 14.6) | 81 ± 3a | 2.71 ± 0.22a | 125 ± 18a | 43.2 ± 0.6b | 95 ± 2b | 92 ± 1c |
| T143A | ∼0.01 (wide) | 3 ± 1c | <0.01c | 1 ± 1c | 22.2 ± 1.6 | 68 ± 5 | 58 ± 5c |
| T143D | 8.61 (7.9, 9.4)a | 102 ± 2 | 0.80 ± 0.01 | 32 ± 4c | 16.3 ± 1.1a | 61 ± 4a | 54 ± 3c |
| T143S | 10.8 (7.8, 17.0) | 106 ± 6 | 2.09 ± 0.18 | 44 ± 5c | 27.7 ± 1.3 | 75 ± 4 | 67 ± 2c |
| T143G | N/D | N/D | N/D | 9 ± 3c | N/D | N/D | N/D |
| T143K | 5.14 (2.7, 45.3) (P = 0.07) | 35 ± 3c | 0.03 ± 0.01c | 8 ± 1c | N/D | N/D | N/D |
| T147A | 10.3 (7.8, 15.0) | 115 ± 5a | 1.79 ± 0.16 | 120 ± 12 | 23.6 ± 0.5 | 93 ± 2 | 91 ± 1c |
| T147D | N/A | <1c | N/A | <1c | <0.1a | N/A | N/A |
| S202A | 11.6 (9.3, 15.1) | 104 ± 4 | 2.74 ± 0.07 | 152 ± 44c | 40.6 ± 2.0 | 89 ± 3 | 83 ± 1a |
| S202D | 11.3 (7.6, 20.9) | 107 ± 8 | 2.48 ± 0.28 | 92 ± 8 | 60.7 ± 3.4 | 95 ± 2 | 86 ± 2b |
| T204A | 12.6 (9.4, 18.9) | 119 ± 7b | 3.67 ± 0.18 | 98 ± 11 | 18.8 ± 0.5 | 91 ± 3 | 89 ± 2b |
| T204D | 6.08 (4.1, 11.2)a | 101 ± 5 | 0.11 ± 0.02 | 69 ± 12 | 39.4 ± 0.2 | 87 ± 1 | 80 ± 2 |
| S208A | 12.7 (10.7, 15.6) | 105 ± 3 | 3.80 ± 0.15 | 93 ± 9 | 22.6 ± 1.5 | 82 ± 6 | 78 ± 2 |
| S208D | 11.3 (9.5, 13.9) | 95 ± 3 | 2.52 ± 0.06 | 96 ± 10 | 56.7 ± 0.4 | 95 ± 1 | 88 ± 2b |
| T209A | 12.1 (7.9, 24.8) | 109 ± 9 | 3.17 ± 0.05 | 101 ± 4 | 32.4 ± 1.5 | 86 ± 4 | 80 ± 2 |
| T209D | 11.9 (9.1, 17.0) | 89 ± 4a | 3.00 ± 0.23 | 87 ± 4 | 20.1 ± 0.4 | 88 ± 2 | 85 ± 1b |
| S280A | 13.3 (10.8, 17.4) | 96 ± 4 | 4.40 ± 0.07 | 70 ± 9a | 50.8 ± 1.0 | 90 ± 2 | 81 ± 2 |
| S280D | 10.4 (9.5, 11.4)a | 69 ± 1c | 1.83 ± 0.02b | 90 ± 10 | <0.1b | 97 ± 1b | ∼100c |
| S294A | 11.5 (8.1, 19.2) | 100 ± 6 | 2.67 ± 0.09 | 75 ± 9 | 36.4 ± 0.5 | 84 ± 2 | 76 ± 2 |
| S294D | 9.1 (5.3, 31.6) | 72 ± 7b | 1.04 ± 0.08a | 76 ± 13 | 49.0 ± 0.5 | 91 ± 1 | 81 ± 1 |
| QQQQ | N/D | 85 ± 3£ | N/D | 33 ± 2£ | 80.5 ± 2.9b | 74 ± 3 | 46 ± 10c |
For cAMP experiments, receptors were transiently transfected in 293/FRT/MRAPβ-cells. Cells were stimulated with 100 nm ACTH in the presence of IBMX for up to 60 min. For internalization experiments, receptors were stably expressed as recombinant 293/FRT/Myc-mutant cell lines and transiently transfected with MRAPβ before stimulation with 100 nm ACTH for up to 60 min. Results are shown as mean ± sem from three to five experiments. &, Determined by nonlinear regression. Data with £ were determined after 15 min of ACTH stimulation in a concentration-response experiment. QQQQ internalization parameters were measured in the same set of experiments as T143A, T143D, and T143S and in parallel with WT. sem assigned with # for WT were estimated based on the means from each experimental group, but statistical significances are based solely on the comparison with WT from a particular experimental group of mutants. The experimental groups are illustrated in Figs. 8 and 9. N/A, Not available; N/D, not determined; R60, cAMP, rate of cAMP accumulation at 60 min; R5, surface, rate of internalization at 5 min.
P < 0.05.
P < 0.01.
P < 0.001.