The agouti-related peptide binds heparan sulfate through segments critical for its orexigenic effects

Abstract

Syndecans potently modulate agouti-related peptide (AgRP) signaling in the central melanocortin system. Through heparan sulfate moieties, syndecans are thought to anchor AgRP near its receptor, enhancing its orexigenic effects. Original work proposed that the N-terminal domain of AgRP facilitates this interaction. However, this is not compatible with evidence that this domain is posttranslationally cleaved. Addressing this long-standing incongruity, we used calorimetry and magnetic resonance to probe interactions of AgRP peptides with glycosaminoglycans, including heparan sulfate. We show that mature, cleaved, C-terminal AgRP, not the N-terminal domain, binds heparan sulfate. NMR shows that the binding site consists of regions distinct from the melanocortin receptor-binding site. Using a library of designed AgRP variants, we find that the strength of the syndecan interaction perfectly tracks orexigenic action. Our data provide compelling evidence that AgRP is a heparan sulfate-binding protein and localizes critical regions in the AgRP structure required for this interaction.

Keywords: AgRP; G protein-coupled receptor (GPCR); glycosaminoglycan; heparan sulfate; heparin-binding protein; mc4r; neuropeptide; syndecan.

Figure 1.

AgRP (83–132) NMR structure (PDB code 1HYK) and schematic indicating ICK and non-ICK regions. The structure of AgRP includes the functional domains shown here and their contribution to MC4R binding. The disulfide bonds are shown in yellow. The schematic highlights the N-terminal segment and C-terminal loop, which are conserved in mammalian sequences. The active loop possesses an RFF triplet (residues 111–113) necessary for melanocortin receptor binding. Positively charged residues within these non-ICK domains are indicated.

Figure 2.

ITC results of size-heterogeneous heparin titrated into solutions of the Cys-rich AgRP(83–132) and the unstructured N-terminal domain. The AgRP Cys-rich domain (AgRP(83–132)) and the flexible N-terminal (N-term) domain (top panel) were synthesized as individual peptides. Heparin was titrated into solutions of each, and thermodynamic parameters, including dissociation constants, were obtained. Although the N-terminal domain exhibited no binding, AgRP(83–132) had a dissociation constant of K_D = 20 ± 6 nm.

Figure 3.

Sequences of AgRP variants used in this study. Mature AgRP(83–132) is displayed first. AgRP(87–120) represents the minimal ICK domain required for high-affinity in vitro binding. Non-ICK positive charges are highlighted in blue, and the removal of positively charged amino acids in AgRP4Q is shown in red.

Figure 4.

Raw isotherms of heparin and heparan sulfate ITC titrations into AgRP peptides. Size-heterogeneous heparin and heparan sulfate were titrated into AgRP variants as indicated. A–C, AgRP(83–132), AgRP(83–120), and AgRP4K isotherms are shown after titration with heparin (left panels) and heparan sulfate (right panels). D, AgRP(87–120), AgRP(87–132), and AgRP4Q isotherms after titration with heparin. As shown, these peptides do not bind heparin and also do not bind heparan sulfate.

Figure 5.

ITC results with AgRP variants. Each AgRP variant was titrated with either size-heterogeneous heparin or heparan sulfate. Thermodynamic data were measured, including dissociation constants and stoichiometry. GAG binding appears to be directly correlated to the non-ICK positive charge. As shown in the % Feeding Above Saline column, taken from our previous rat feeding study, this trend correlates with 24-h feeding. Four ITC experiments were repeated with each peptide two to four times, and the reported error is the standard deviation of each set of measurements. Feeding data are presented as percent feeding above saline and standard deviation.

Figure 6.

HSQC chemical shift perturbation data from titration of heparin into AgRP peptides. A, top panels, HSQC data of AgRP(83–132) titrated with heparin dp6 showing residues from both C-terminal loop (Lys-121, Leu-122, and Ala-125) and N-terminal segment (Arg-86) shifting. A and B, bottom panels, data plotted as Δ chemical shift. A Δδ value is considered significant when it exceeds 1 S.D. over the mean chemical shift. Non-ICK regions, which are important for GAG binding by ITC, are boxed. B, top panel, HSQC data of AgRP4K titrated with heparin dp2. Residues from both the C-terminal loop (Lys-121, Leu-122, Lys-123, and Lys-125) and N-terminal segment (Arg-85 and Arg-86) shift systematically with added dp2. Non-ICK regions are boxed. We were unable to observe Lys-83 and Lys-84 because of solvent exchange.

Figure 7.

Comparison of GAG binding and electrostatic surfaces of AgRP peptides. A, top panel, NMR chemical shift perturbation data from heparin dp6 titration depicted on the surface of AgRP(83–132). Residues that shift above the average chemical shift and 1 S.D. are colored as noted. Bottom panel, the same perspective of AgRP(83–132), with surface electrostatics as calculated by APBS. This comparison shows similarities in the most positively charged regions of the peptide to regions perturbed by heparin titration. The dashed red ovals highlight the non-ICK segments and the region between them, and the RFF triplet is indicated by solid red ovals. B, top panel, NMR chemical shift perturbation data from heparin dp2 titration depicted on the surface of AgRP4K with the same color code as in A. Bottom panel, the same perspective of AgRP4K, with surface electrostatics as calculated by APBS.

Figure 8.

Schematic of a proposed mechanism for MC4R modulation by syndecans. Full-length AgRP is posttranslationally cleaved to release AgRP(83–132). This mature form of AgRP (represented here) interacts with both heparan sulfate proteoglycans, via GAGs, and MC4R. The positively charged non-ICK regions of the peptide are dispensable for MC4R binding and activity but required for high-affinity GAG binding. Our results suggest that syndecans either concentrate AgRP near its receptor or act as a co-receptor.