Research involving human subjects was approved by the BloodCenter of Wisconsin Institutional Review Board, and all human participants gave written informed consent in accordance with the Declaration of Helsinki. Results Structure of the PECAM-1 cytoplasmic domain in the presence and absence of DPC micelles We used an NMR approach to investigate the structural properties of the PECAM-1 cytoplasmic domain. propose a novel model for regulation of inhibitory signaling by ITIM-containing receptors that relies on reversible plasma membrane interactions and sequential ITIM phosphorylation. Introduction Immunoreceptor tyrosine-based inhibitory motif (ITIM)Ccontaining receptors oppose cellular activation by receptors that are coupled to immunoreceptor tyrosine based-activation motif (ITAM)Ccontaining subunits.1 The inhibitory function of ITIM-containing receptors depends on Src homology (SH) 2 domainCdependent binding of tyrosine or inositol phosphatases to phosphorylated ITIMs within the cytoplasmic domain and subsequent dephosphorylation of signal transduction pathway components that are required for ITAM-dependent cellular activation.1 Src family kinases initiate signaling by both ITIM- and ITAM-containing receptors; however, the mechanisms that control access of ITIM or ITAM sequences to active Src family kinases are not well understood. Recent studies have revealed that the ITAMs of T-cell receptorCassociated signaling subunits, which are intrinsically unstructured in an aqueous environment, exhibit increased -helical content and decreased phosphorylation on interaction with detergent or phospholipid vesicles that mimic the plasma membrane.2C4 These findings suggested that signal transduction by ITAM-containing receptors is regulated by reversible membrane association. The extent to which this principle applies to ITIM-containing receptors has not been determined. The ITIM-containing receptor family encompasses a large number of different Ig-domainCcontaining and C-type lectin receptors that are expressed on an array of cells of hematopoietic origin.1 Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) is a member of the Ig-ITIM subfamily of ITIM-containing receptors5 that is expressed on all endothelial cells and on most hematopoietic cells, including platelets, mast cells, lymphocytes, and monocytes.6 PECAM-1 is a 130-kDa type I transmembrane glycoprotein that contains 6 extracellular Ig homology domains, a single-pass transmembrane region, and a long cytoplasmic domain that contains 2 ITIMs surrounding tyrosine (Y) residues that are found at positions 663 and 686 within the mature form of human PECAM-1, the phosphorylation of which supports recruitment and activation of Src homology 2 domainCcontaining protein tyrosine phosphatases, including SHP-2 and, to a lesser extent, SHP-1.7 Formation of PECAM-1/SHP-2 complexes is associated with inhibition of platelet, mast F2RL3 cell, and lymphocyte activation via ITAM-coupled receptors.7 Sofosbuvir impurity C As with other ITIM-containing Sofosbuvir impurity C receptors, Src family kinases are involved in PECAM-1 tyrosine phosphorylation7; however, the mechanisms that control phosphorylation of the PECAM-1 ITIMs are not known. In the present study, we determined the structure of the ITIM-containing PECAM-1 cytoplasmic domain, in the presence and absence of detergent micelles to mimic the plasma membrane. We found that the PECAM-1 cytoplasmic domain was intrinsically unstructured in aqueous solution but that a localized region of it adopted -helical conformation on interaction with a plasma membrane mimetic. The membrane-interacting portion of the PECAM-1 cytoplasmic domain encompassed the C-terminal ITIM, which became preferentially phosphorylated relative to the nonCmembrane-interacting N-terminal ITIM, as well as a serine residue that was susceptible to inducible phosphorylation. On the basis of these results, we conclude that plasma membrane interactions control access of cytoplasmic ITIM and serine residues to the kinases that phosphorylate them. Methods NMR spectroscopy Generation of the recombinant proteins that encompassed the entire (PECAM594-711) or the C-terminal half (PECAM659-711) of the PECAM-1 cytoplasmic domain is described in supplemental Methods (available on the Web site; see the Supplemental Materials link at the top of the online article). A two-dimensional Sofosbuvir impurity C 15N,1H heteronuclear single quantum coherence (HSQC) titration experiment was performed with a sample that contained 600M 15N-labeled PECAM594-711 in nuclear magnetic resonance (NMR) buffer that contained 90% H2O/10% D2O. For dodecylphosphocholine (DPC) dose-response studies, 600mM DPC was added after the first spectrum was collected, and buffer that contained 600M PECAM594-711 was added for each subsequent titration point. Paramagnetic broadening was measured by acquiring 2-dimensional 1H-13C HSQC spectra on PECAM659-711 with 600 mM DPC in the presence and absence of 0.5 mM MnCl2. Three-dimensional (3D) heteronuclear backbone experiments and heteronuclear 15N-1H nuclear Overhauser effect (hetNOE) experiments8 were performed as described previously9 with samples that contained 1.25mM 13C/15N-labeled PECAM594-711 or PECAM659-711 with or without 600mM DPC in the same buffer. NMR data were acquired at 25C on a Bruker 500-MHz spectrometer equipped with a triple-resonance CryoProbe and processed with NMRPipe software.10 Sofosbuvir impurity C Initial 1H, 15N, and 13C resonance assignments for PECAM594-711 (+DPC) were obtained from the PINE-NMR server11 (version 1.0; http://miranda.nmrfam.wisc.edu/PINE/), with peak lists from 3D HNCO,.