AgRP(83–132) is obtained by cleavage of the oxidatively folded full-length protein.
(A) Inhibition of NDP-MSH-stimulated cAMP generation at hMC4R by full-length AgRP, and corresponding Schild analysis, reveals competitive antagonism with a dissociation constant (K_i) of 3.6 nM.
(B) Both full-length AgRP and AgRP(83–132) inhibit MC4R cAMP production (measured at antagonist concentrations of 0.01 μM and 0.1 μM) stimulated by α-MSH, but AgRP(83–132) is substantially more potent and, to within experimental error, equivalent to AgRP(86–132) with a K_i of 0.36 nM.
(C) Displacement of the radioligand ¹²⁵I-NDP-MSH from MC4R by full-length AgRP and C-terminal AgRP, giving respective IC₅₀ values of 18.1 ± 0.4 nM and 1.6 ± 0.2 nM.
Error bars represent standard deviations determined from triplicate measurements.

(A) The measured ¹H chemical shifts from AgRP(83–132), minus the consensus random coil chemical shifts. The significant variation is consistent with a folded domain. Circles represent measured values; squares represent proline, which is not observable in the HSQC spectra. The arrows show the locations of β strands adjacent to the RFF triplet (residues 111–113).
(B) The difference in chemical shifts, full-length minus C-terminal, for residues 83–132. Note that the vertical axis is expanded by a factor of three compared to that in (A). The limited scatter demonstrates that the HSQC from residues 83–132 in full-length AgRP is essentially equivalent to that of the isolated C-terminal domain, AgRP(83–132). The 2 residues that do show significant ¹H chemical shift variations are Arg89 and His91, as indicated.

(A) [¹⁵N, ¹H]-HSQC spectrum of full-length MKd5-AgRP. Assigned peaks are from residues 83–132.
(B) ¹⁵N [¹H]-NOE spectrum of full-length MKd5-AgRP. Positive NOEs are red, and negative NOEs are green. Almost all N-terminal residues are either negative or unobservable, demonstrating the flexibility of this segment.

(A) Representation of full-length AgRP. The structure of the Cys-rich C-terminal domain was determined previously by ¹H NMR (PDB code: 1HYK) and is preserved in the full-length protein. Positive ¹⁵N[¹H]-NOEs from residues 87–130 (Figure 3B) show that this domain tumbles with a correlation time (τ_c) greater than 1 ns, consistent with a structured unit. By contrast, the N-terminal domain is flexible and has a backbone τ_c ≪ 1 ns. Cysteine residues involved in disulfide bonds are yellow; residues in the RFF triplet, required for high-affinity MCR interactions, are blue. Residues in the putative proprotein convertase recognition site are red. Cleavage takes place after Arg82. The demonstrated flexibility of this segment is consistent with PC cleavage in vivo. CYANA calculations [32] produced a broad ensemble of possible N-terminal structures (500 total were calculated); for clarity, only 1 of the several lowest energy structures is shown.
(B) The NMR structure of the C-terminal domain of ASIP.
(C) Structural alignment of ASIP with the corresponding domain of AgRP [33].

Relative evolutionary constraint for each group of indicated proteins was carried out as described in Experimental Procedures; the multiple sequence alignments are indicated below each plot. Areas highlighted in blue and red indicate regions of high local constraint that correspond to the N-terminal domain of ASIP or the C-terminal domains of both proteins, respectively. Accession numbers for ASIP orthologs (and their species and source) are P42127 (human, UniProt), Q5UK76 (dog, UniProt), Q29414 (cow, UniProt), Q6ZYM3 (pig, UniProt), Q03288 (mouse, UniProt), Q99JA2 (rat, Uniprot), ENSMODP00000003361 (opossum, Ensembl), ENSGALP00000034003 (chicken, Ensembl), and Q5CC35 (gold-fish, Uniprot). Accession numbers for AgRP orthologs (and their species and source) are O00253 (human, UniProt), 73957497 (dog, GenBank), P56413 (cow, Uniprot), Q9TU18 (pig, UniProt), P56473 (mouse, UniProt), 62665164 (rat, GenBank), ENSMODP00000007302 (opossum, Ensembl), and ENSGALP00000003505 (chicken, Ensembl).