Abstract
The interaction between the short 88Ser-Arg-Ser-Arg-Tyr92 sequence of the urokinase receptor (uPAR) and the formyl peptide receptor type 1 (FPR1) elicits cell migration. We generated the Ac-(D)-Tyr-(D)-Arg-Aib-(D)-Arg-NH2 (RI-3) peptide which inhibits the uPAR/FPR1 interaction, reducing migration of FPR1 expressing cells toward N-formyl-methionyl-leucyl-phenylalanine (fMLF) and Ser-Arg-Ser-Arg-Tyr (SRSRY) peptides. To understand the structural basis of the RI-3 inhibitory effects, the FPR1/fMLF, FPR1/SRSRY and FPR1/RI-3 complexes were modeled and analyzed, focusing on the binding pocket of FPR1 and the interaction between the amino acids that signal to the FPR1 C-terminal loop. We found that RI-3 shares the same binding site of fMLF and SRSRY on FPR1. However, while fMLF and SRSRY display the same agonist activation signature (i.e. the series of contacts that transmit the conformational transition throughout the complex), translating binding into signaling, RI-3 does not interact with the activation region of FPR1 and hence does not activate signaling. Indeed, fluorescein-conjugated RI-3 prevents either fMLF and SRSRY uptake on FPR1 without triggering FPR1 internalization and cell motility in the absence of any stimulus. Collectively, our data show that RI-3 is a true FPR1 antagonist and suggest a pharmacophore model useful for development of compounds that selectively inhibit the uPAR-triggered, FPR1-mediated cell migration.
Highlights
Cell migration is a sequential and interrelated multistep process that regulates physiological processes such as embryonic development, tissue repair and immune-cell trafficking in both embryo and adult tissues[1]
It is important to highlight that: i) the binding site, that is “cone-shaped with the tip towards the trans-membrane region”, is small and can accommodate only one peptide at a time considering that its radius and its height are lower than the length of the peptides; ii) the charged and aromatic residues in the pocket allow the charged and aromatic residues of the peptides to strongly interact with formyl peptide receptor type 1 (FPR1) by ionic-aromatic and stacking interactions, H-bonds, and salt bridges
Cell migration offers rich targets for intervention since its control may improve pathologic conditions sustained by an altered cell motility, including chronic inflammation and neoplastic diseases
Summary
Cell migration is a sequential and interrelated multistep process that regulates physiological processes such as embryonic development, tissue repair and immune-cell trafficking in both embryo and adult tissues[1]. Besides focusing proteolytic activity of uPA on the cell membrane[5], uPAR, upon binding to uPA, initiates in a protease independent manner the intracellular signaling pathways that regulate physiologic processes such as wound repair and immune responses, as well as pathologic conditions such as inflammation and tumor metastases[4,6]. We and others documented that the uPAR84-95 sequence as well as the synthetic shorter pentapeptide uPAR88-92(Ser-Arg-Ser-Arg-Tyr, SRSRY) elicit chemotaxis and promote directional cell migration and angiogenesis in vitro and in vivo[12,13,14]. We proven that both uPAR84-95 and SRSRY exert chemotactic activity by interacting with the formyl peptide receptor type 1 (FPR1) which, in turn, internalizes. The cyclic [SRSRY] peptide competes with both SRSRY and N-formylmethionyl-leucyl-phenylalanine (fMLF) peptides for binding to FPR1 and inhibits FPR1activation and internalization, causing a dramatic reduction of monocyte recruitment into inflamedtissues[17,18,19]
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