Individually Distinct Ig Homology Domains in PECAM-1 Regulate Homophilic Binding and Modulate Receptor Affinity

doi:10.1074/jbc.271.19.11090

Individually Distinct Ig Homology Domains in PECAM-1 Regulate Homophilic Binding and Modulate Receptor Affinity (*)

From the (1)Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53233-2194, the
(2)Department of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104,
(3)Amgen Boulder, Inc., Boulder, Colorado 80301, and the
(4)Departments of Cellular Biology and Pharmacology, The Medical College of Wisconsin, Milwaukee, Wisconsin 53226

^¶ To whom correspondence should be addressed:
Blood Research Institute, The Blood Center of Southeastern Wisconsin, 638 N. 18th St., Milwaukee, WI 53233-2194.
Tel.: 414-937-6237; Fax: 414-937-6284.

Abstract

PECAM-1 (CD31) is a 130-kDa member of the immunoglobulin (Ig) gene superfamily that is constitutively expressed at high concentration at endothelial cell intercellular junctions and at moderate density on the surface of circulating leukocytes and platelets. Recent in vitro and in vivo studies have shown that PECAM-1 plays a central role in mediating the extravasation of leukocytes from the vessel wall in response to inflammatory mediators. To study the binding characteristics of PECAM-1, phospholipid vesicles were prepared and examined by flow cytometry and immunofluorescence microscopy for their ability to associate with each other and with cells. Proteoliposomes containing high concentrations of PECAM-1 interacted homophilically with each other, forming large self-aggregates. PECAM-1 proteoliposomes, as well as soluble bivalent PECAM-1 in the form of a PECAM-1/IgG immunoadhesin, associated homophilically with cells expressing human, but not murine, PECAM-1. This binding could be completely inhibited by monoclonal antibody Fab fragments specific for Ig homology Domain 1 or Domains 1 + 2. Binding studies using cells expressing human PECAM-1 deletion mutants and murine/human chimeras confirmed that both Ig Domains 1 and 2 were both necessary and sufficient for homophilic binding. In contrast, engagement of membrane-proximal Domain 6 with monoclonal antibody Fab fragments had the opposite effect and augmented the binding of PECAM-1 proteoliposomes to cells. Thus, PECAM-1, like certain integrins, appears to be capable of antibody-induced conformational changes that alter affinity for its ligand. Similar changes induced by physiologic stimuli could be important in regulating the function of PECAM-1 in vascular cells.

Footnotes

↵^§ Established Investigators of the American Heart Association.
↵* This work was supported by Grants HL-40926 (to P. J. N.), HL-43611 (to S. M. A.), and HL-03382 (to H. M. D.) from the National Institutes of Health and by a Robert Wood Johnson Foundation Minority Faculty grant (to H. M. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PAGE

polyacrylamide gel electrophoresis

HUVEC

human umbilical vein endothelial cell

FBS

fetal bovine serum

HBSS

Hanks' balanced salt solution

GP

glycoprotein

TBS

Tris-buffered normal saline

PC

phosphatidylcholine

NBD

7-nitrobenzo-2-oxa-1,3-diazole

CHO

Chinese hamster ovary

LAK

lymphokine-activated killer

FITC

fluorescein isothiocyanate.
↵²C. Paddock and P. J. Newman, unpublished observations.
- Received February 6, 1996.