3.1 Expression and signaling of chimeric receptors
We prepared chimeric receptors in which segments of the human MC2
receptor, which relies on the accessory protein MRAP for trafficking and signaling, were replaced with corresponding sequences from the human MC4
receptor, which is able to function without MRAP. We tagged all receptors with the HA epitope at the amino-terminus to facilitate quantification of receptor on the plasma membrane. Receptors were transiently expressed in CHO cells together with MRAP or either RAMP3 (an accessory protein for several class B receptors that does not affect melanocortin receptors)(Parameswaran and Spielman, 2006
) or GFP as controls.
To measure receptors on the plasma membrane, we used a protocol that has been carefully validated previously(Jones et al., 2007
; Sebag and Hinkle, 2007
). Intact cells were incubated with monoclonal anti-HA antibodies, washed to remove free antibody, and then fixed and signal quantified by standard ELISA protocols. To measure receptor signaling, we either quantified cAMP directly or used a reporter assay. In the direct assay, cells were incubated with or without ACTH or NDP-α-MSH in the presence of a phosphodiesterase inhibitor for 30 min when the cAMP concentration was determined. In the reporter assay, cells were transfected with cDNA encoding luciferase driven by multiple copies of a cAMP-dependent cAMP response element (CRE) from the rat insulin promoter (CRE-luciferase) (Chepurny and Holz, 2007
) together with receptors and MRAP, then incubated with or without agonist or forskolin for 5 h, when luciferase activity was measured. Forskolin stimulates adenylyl cyclase directly. The reporter assay is more sensitive and was useful in studying chimeric receptors with weak signaling. In cells expressing MC2
receptor and MRAP, the EC50
for ACTH was approximately 10−10
M in the CRE-luciferase assay and over 20-fold higher, 2.4 ×10−9
M, in the direct cAMP assay, highlighting the difference in sensitivity.
receptor was expressed poorly on the plasma membrane in the absence of MRAP and strongly in the presence of the accessory protein (). ACTH-dependent cAMP generation by the MC2
receptor was highly dependent on MRAP (). In contrast, MRAP decreased surface expression of the MC4
receptor with little effect on cAMP response. The ability of MRAP to inhibit MC4
receptor expression was also observed by Chan et al.(Chan et al., 2009
). In our experiments, MRAP did not have a significant effect on MC4
receptor signaling. To determine whether the same concentrations of MRAP were required to affect MC2
receptors, we transfected cells with different amounts of MRAP cDNA and measured receptor expression (). MRAP increased MC2
receptor on the cell membrane and inhibited MC4
receptor trafficking at the same concentrations. Changing the ratio of MRAP to MC2
receptor did not change the EC50
for ACTH response (data not shown).
Fig. 1 Effects of MRAP on MC2 and MC4 receptors. Cells were transfected with plasmids encoding MC2 receptor (MC2R), MC4 receptor (MC4R) and either RAMP3 (Control) or MRAP. (A and C) Surface expression of receptors was measured by ELISA as described under Methods (more ...)
Receptor constructs are shown schematically in the figures, and sequence details are provided in Supplemental Fig. 1
. Signaling by chimeric receptors was tested in both direct cAMP assays (shown in Supplemental Fig. 2
) and in CRE-luciferase reporter assays (shown in and for receptors that are primarily MC2
, respectively). To be sure that all chimeric receptors would be activated regardless of ligand specificity, we challenged cells with a combination of 1 µM ACTH and 1 µM NDP-α-MSH in the reporter assays.
In general, MRAP promoted the trafficking of the chimeras that are predominantly MC2 and reduced cell surface expression of those that are predominantly MC4. A few of the chimeric receptors were not expressed well in either case. The MC2-like chimeric receptors either required MRAP for signaling or failed to respond to ACTH, with the exception of chimera 2C5 (). The MC4-like receptors that were capable of signaling did not require MRAP, with the exception of the 4C4 receptor. MRAP enhanced signaling by the 4C4 chimera, which contains the last two transmembrane domains, third extracellular loop (ECL3) and C-tail of the MC2 receptor. MRAP inhibited surface expression of the 4C4 chimera and the MC4 receptor parent. The converse receptor, 2C4, with a C-terminus from the MC4 receptor, did not signal. A number of the chimeric receptors were present at high levels on the plasma membrane but failed to give any cAMP response (most notably 2C1 and 4C2), whereas the 2C2 receptor expressed at low levels but signaled strongly.
One clear finding was the presence of significant levels of the 2C1 chimera on the cell surface in the absence of MRAP. The 2C1 receptor has the N-terminus, first transmembrane domain (TM1) and first intracellular loop (ICL1) from the MC4
receptor. To verify the ELISA result, we expressed the parent MC2
receptor and the 2C1 chimera with and without MRAP and stained for surface receptor using fluorescence microscopy. As shown in , the 2C1 receptor was localized on the plasma membrane with or without MRAP. In contrast, the MC2
receptor was visible only in cells co-expressing MRAP. We narrowed the region responsible for MRAP-independent trafficking by generating mutants containing only TM1, TM1 and ICL-1, or only ICL-1 from the MC4
receptor. Replacing TM1 of the MC2
receptor with the corresponding transmembrane segment from MC4
was sufficient to allow the receptor to undergo trafficking to the cell surface even in the absence of MRAP (, 2C1b). Despite substantial surface expression, the 2C1 chimera gave essentially no cAMP or CRE-luciferase response with or without the accessory protein (Supplemental Fig. 2
, and ). Signaling results were remarkably different for the derivatives of 2C1. Simply replacing the extracellular N-terminal region of the 2C1 receptor with the native MC2
receptor sequence (chimera 2C1a) restored strong signaling. The receptor containing only TM1 from the MC4
receptor (chimera 2C1b) gave a very strong ACTH response that was highly dependent on MRAP, as did receptor 2C1c with only ICL1 from the MC4
Fig. 4 Surface expression of MC2 and 2C1 receptors. Cells were transfected with plasmids encoding GFP and either MC2 receptor or 2C1 chimeric receptor together with either control plasmid or MRAP. Live cells were incubated with monoclonal anti-HA antibody and (more ...)
Fig. 5 Importance of amino-terminal regions of MC2 receptor. (A) Amino acid sequences of receptor constructs, beginning at amino acid 2 after the HA tag (MYPYDVPDYAYPYDVPDYA). MC2 receptor sequences are shown in red, MC4 in blue. (B) Cells were transfected with (more ...)
These results raise the possibility that N-terminal regions of the MC2
receptor are responsible for the requirement for MRAP for proper trafficking. We attempted to learn whether these domains of the MC2
receptor were sufficient to confer MRAP dependence to the MC4
receptor by studying the converse of the 2C1 receptor, 4C1 (). The 4C1 receptor was expressed very poorly on the plasma membrane and gave a small but significant response in the CRE-luciferase system that was the same with or without MRAP. Signaling by the 4C1 chimera was too weak to detect in cAMP assay (Supplemental Fig. 2
3.2 Ligand specificity of chimeric receptors
The 2C2 chimera, which has TMs 2 and 3 and the connecting first extracellular loop (ECL-1) from the MC4
receptor, showed low, MRAP-enhanced surface expression but striking agonist responses in both the CRE-luciferase () and direct cAMP assays (Supplemental Fig. 2
). The 2C2 receptor also gave a significant response in the sensitive reporter assay in the absence of MRAP (). We also tested the ability the chimeric receptors to respond to ACTH or NDP-α-MSH individually. Unexpectedly, we found that the 2C2 chimera responded to NDP-α-MSH as well as ACTH and displayed substantial constitutive activity in the CRE-luciferase assay (). These properties resemble those of the MC4
Fig. 6 Importance of regions from TM2 through TM3 of the MC2 receptor. (A) Amino acid sequences of receptor constructs. MC2 receptor sequences are shown in red, MC4 in blue. (B) Cells were transfected with plasmids encoding receptors and either control plasmid (more ...)
Complete concentration-response curves for ACTH and NDP-α-MSH are shown in . Cells transfected with the 2C2 chimera and MRAP responded to both ACTH (EC50 = 37 nM) and NDP-α-MSH (EC50 = 7.2 nM). The MC4 receptor is much less sensitive to ACTH than the MC2 receptor. Likewise, the 2C2 chimera was 380-fold less sensitive to ACTH than the MC2 receptor (EC50 = 37 vs. 0.097 nM). The 2C2 receptor was not as sensitive to NDP-α-MSH as the MC4 receptor, which has an EC50 of 0.011 nM for NDP-α-MSH and an EC50 of 1.3 nM for ACTH in the CRE-luciferase assay. The substantial constitutive activity of 2C2 was entirely MRAP-dependent.
Fig. 7 Ligand specificity of MC2, MC4, 2C2 and 2C3 receptors. Cells were transfected with CRE-luciferase, receptors and MRAP or control plasmid. The next day cells were incubated with vehicle, 20 µM forskolin or different concentrations of ACTH or NDP-α-MSH (more ...)
When it was expressed without MRAP, the 2C2 receptor responded to NDP-α-MSH but the potency was relatively low (EC50 = 57 nM). The maximal responses of the 2C2 receptor to NDP-α-MSH were approximately the same with or without the accessory protein. On the other hand, ACTH elicited no significant response via the 2C2 receptor expressed without MRAP.
To dissect the basis for the unexpectedly strong signaling of the 2C2 chimera, we engineered receptors that contained either ECL1 from the MC4 receptor (2C2a), ECL1 plus TM2 (2C2b), or ECL1 plus TM3 (2C2c). Two of these receptors trafficked well to the cell surface with MRAP but displayed very low signaling (), while the third did not express or signal.
The 2C3 chimeric receptor, which has TMs 4 and 5 and the connecting ICL-2 loop from the MC4
receptor, also expressed and signaled well ( and Supplemental Fig. 2
). The 2C3 receptor resembled the MC2
receptor, requiring MRAP for trafficking and signaling and responding to low concentrations of ACTH (EC50
= 0.59 nM) but not to NDP-α-MSH (). The 2C3 receptor exhibited weak constitutive activity.