Hypothetical model of polyP-mediated proinflammatory nuclear cytokines (HMGB1 or H4) signaling. The 2 nuclear cytokines (only HMGB1 used in the graph) bind to the positively charged N-terminus variable domain (V) of RAGE receptor to elicit proinflammatory signaling responses in endothelial cells (or cells of the innate immune system). In the case of HMGB1, this can occur through the acidic C-terminus of the protein (shown in the model as a minus sign). However, relatively high concentrations of nuclear cytokines are required to initiate signaling because both HMGB1 and H4 are also highly positively charged. The interaction of the acidic polyP polymers with positively charged residues of either nuclear protein neutralizes the basic charges of these residues, thereby eliminating their repulsive interactions with the positively charged ligand-binding domain of RAGE (V) and enhancing their affinity for the receptor. Depending on its polymer size, polyP can bind to multiple nuclear cytokines (ie, polyP-700 shown in the model), thus simultaneously activating multiple receptors. Because polyP can also bind to P2Y₁, this interaction is further enhanced by a bridging mechanism. The polyP-loaded ligand interaction with RAGE results in the clustering of the receptor and the P2Y₁ bridging stabilizes oligomeric forms of RAGE, thus facilitating the cooperative interaction of the cytoplasmic tails of the receptor with the adaptor molecule mDia1, which is responsible for initiating downstream signaling. In the absence (or limiting concentration) of nuclear cytokines, polyP itself can also bind to either RAGE or P2Y₁ separately or to both receptors by a bridging mechanism to initiate inflammatory signaling responses (not shown). V, C1, and C2 represent the N-terminal variable region followed by constant regions 1 and 2 of the extracellular domain of RAGE.