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J. Biol. Chem., Vol. 282, Issue 3, 1805-1818, January 19, 2007

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Phylogenetic Divergence of CD47 Interactions with Human Signal Regulatory Protein Reveals Locus of Species Specificity

IMPLICATIONS FOR THE BINDING SITE^*

From the Biophysical Engineering Laboratory, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Cell-cell interactions between ubiquitously expressed integrin-associated protein (CD47) and its counterreceptor signal regulatory protein (SIRP) on phagocytes regulate a wide range of adhesive signaling processes, including the inhibition of phagocytosis as documented in mice. We show that CD47-SIRP binding interactions are different between mice and humans, and we exploit phylogenetic divergence to identify the species-specific binding locus on the immunoglobulin domain of human CD47. All of the studies are conducted in the physiological context of membrane protein display on Chinese hamster ovary (CHO) cells. Novel quantitative flow cytometry analyses with CD47-green fluorescent protein and soluble human SIRP as a probe show that neither human CD47 nor SIRP requires glycosylation for interaction. Human CD47-expressing CHO cells spread rapidly on SIRP-coated glass surfaces, correlating well with the spreading of primary human T cells. In contrast, CHO cells expressing mouse CD47 spread minimally and show equally weak binding to soluble human SIRP. Further phylogenetic analyses and multisite substitutions of the CD47 Ig domain show that human to cow mutation of a cluster of seven residues on adjacent strands near the middle of the domain decreases the association constant for human SIRP to about one-third that of human CD47. Direct tests of cell-cell adhesion between human monocytes and CD47-displaying CHO cells affirm the species specificity as well as the importance of the newly identified binding locus in cell-cell interactions.

Received for publication, April 24, 2006 , and in revised form, October 10, 2006.

^* This work was supported by National Institutes of Health Grants R21 and R01 (to D. E. D.), a National Science Foundation career grant (to E. T. B.), and a predoctoral fellowship from Merck (to S. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.

¹ To whom correspondence should be addressed: Molecular and Cell Biophysics Laboratory, University of Pennsylvania, 112 Towne Bldg., 220 S. 33rd St., Philadelphia, PA 19104. Tel.: 215-898-4809; Fax: 215-573-2093; E-mail: discher{at}seas.upenn.edu.

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