Central to the ability of vascular cells to adhere to the extracellular matrix and to each other is an abundant supply of cell surface adhesion molecules that, in addition to influencing the adhesive phenotype of the cell, are also capable of transmitting signals into, and responding to signals from, the cell interior. Such post-ligand events occur by virtue of the ability of these transmembrane proteins to interact with intracellula kinases and phosphatases, G-proteins, adapter proteins, and cytoskeletal components. PECAM-1 (also known as CD31) is a cellular adhesion and signaling receptor comprised of six extracellular immunoglobulin (Ig)-like homology domains, a short transmembrane domain, and a 118 amino acid cytoplasmic domain that becomes serine and tyrosine phosphorylated upon cellular activation. PECAM-1 expression is restricted to blood and vascular cells. In circulating platelets and leukocytes, PECAM-1 functions largely as an inhibitory receptor that, via regulated sequential phosphorylation of its cytoplasmic domain, limits cellular activation responses. PECAM-1 is also highly expressed at endothelial cell intercellular junctions, where it functions as a mechanosensor, as a regulator of leukocyte trafficking, and in the maintenance of endothelial cell junctional integrity. Previous studies have shown that PECAM-1-PECAM-1 homophilic interactions mediated by N-terminal Ig domain 1 are required for border localization, and contribute importantly not only to steady-state endothelial cell barrier stability, but also to recovery of endothelial cell junctional integrity, both in vitro and in vivo, following inflammator or hemostatic challenge. The overall goal of this competitive renewal application is to build on recent progress in the field of PECAM-1 biology to address outstanding, broadly applicable, and scientifically-important questions in the fields of cell adhesion and regulation.
Two Specific Aims will focus on novel mechanisms that regulate the homophilic binding affinity of PECAM-1, while a third Specific Aim will deal with the cellular mechanisms of control over PECAM-1 phosphorylation that impose tight, temporal regulation of its inhibitory function. Specifically, ovr the next five-year period we propose to (1) Examine the structural and functional basis of allosteric regulation of PECAM-1 homophilic binding affinity, (2) Identify serine/threonine kinase(s) that trigger PECAM-1-mediated inhibitory signaling, and (3) Define atomic-level structural determinants of PECAM-1-mediated homophilic interactions that account for its ability to regulate and maintain endothelial cell junctional integrity. Together, these studies comprise a coordinated, focused research program designed to improve our understanding of the function of this novel vascular cell adhesion and signaling molecule in the blood and vascular cells in which it is expressed. Many of the principles established in this proposal, including affinity modulation within the Ig superfamily and the regulation of ITIM-mediated inhibitory signaling by sequential phosphorylation, should be impactful well beyond the field of PECAM-1 biology.

Public Health Relevance

PECAM-1 is a 130 kDa member of the Immunoglobulin gene superfamily that is expressed on the surface of circulating platelets and leukocytes, and is also a major constituent of the endothelial cell intercellular junction; where up to 2 x 106 molecules per cell are expressed. Owing in part to its unique tissue distribution, and to its role as a vascular cell adhesion and signaling receptor, PECAM-1 has been shown to function prominently in thrombosis, hemostasis, and inflammatory disorders. This proposal seeks to understand how the adhesive properties of PECAM-1 are regulated to support the integrity of the vascular endothelium, and how the cytoplasmic domain functions to inhibit cellular activation responses.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL040926-29
Application #
9177763
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Kindzelski, Andrei L
Project Start
1988-07-01
Project End
2018-11-30
Budget Start
2016-12-01
Budget End
2017-11-30
Support Year
29
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Bloodcenter of Wisconsin, Inc.
Department
Type
DUNS #
057163172
City
Milwaukee
State
WI
Country
United States
Zip Code
53233
Paddock, Cathy; Zhou, Dongwen; Lertkiatmongkol, Panida et al. (2016) Structural basis for PECAM-1 homophilic binding. Blood 127:1052-61
Lertkiatmongkol, Panida; Liao, Danying; Mei, Heng et al. (2016) Endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31). Curr Opin Hematol 23:253-9
Newman, Debra K; Fu, Guoping; Adams, Tamara et al. (2016) The adhesion molecule PECAM-1 enhances the TGF-?-mediated inhibition of T cell function. Sci Signal 9:ra27
Privratsky, Jamie R; Newman, Peter J (2014) PECAM-1: regulator of endothelial junctional integrity. Cell Tissue Res 355:607-19
Mei, Heng; Campbell, Jay M; Paddock, Cathy M et al. (2014) Regulation of endothelial cell barrier function by antibody-driven affinity modulation of platelet endothelial cell adhesion molecule-1 (PECAM-1). J Biol Chem 289:20836-44
Tourdot, Benjamin E; Brenner, Michelle K; Keough, Kathleen C et al. (2013) Immunoreceptor tyrosine-based inhibitory motif (ITIM)-mediated inhibitory signaling is regulated by sequential phosphorylation mediated by distinct nonreceptor tyrosine kinases: a case study involving PECAM-1. Biochemistry 52:2597-608
Kuckleburg, Christopher J; Newman, Peter J (2013) Neutrophil proteinase 3 acts on protease-activated receptor-2 to enhance vascular endothelial cell barrier function. Arterioscler Thromb Vasc Biol 33:275-84
Privratsky, Jamie R; Tilkens, Sarah B; Newman, Debra K et al. (2012) PECAM-1 dampens cytokine levels during LPS-induced endotoxemia by regulating leukocyte trafficking. Life Sci 90:177-84
Kuckleburg, Christopher J; Tilkens, Sarah B; Santoso, Sentot et al. (2012) Proteinase 3 contributes to transendothelial migration of NB1-positive neutrophils. J Immunol 188:2419-26
Paddock, Cathy; Lytle, Betsy L; Peterson, Francis C et al. (2011) Residues within a lipid-associated segment of the PECAM-1 cytoplasmic domain are susceptible to inducible, sequential phosphorylation. Blood 117:6012-23

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