Two Distinct Cell Attachment Sites in Entactin Are Revealed by Amino Acid Substitutions and Deletion of the RGD Sequence in the Cysteine-rich Epidermal Growth Factor Repeat 2 (*)
- From the (1)Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
- § To whom correspondence should be addressed. Tel.: 412-624-4304; Fax: 412-624-4759.
Abstract
The basement membrane glycoprotein, entactin, has previously been shown to promote cell attachment and chemotaxis. We have
constructed a panel of glutathione S-transferase fusion proteins that encompasses the four major structural domains of entactin, G1, G2, E, and G3. These proteins
have been synthesized in bacteria and purified by affinity chromatography. The connecting stalk of entactin, E, which contains
four cysteine-rich EGF homology repeats and the integrin receptor RGD recognition sequence, has been modified by deletion
of the RGD sequence and substituting glutamic acid for aspartic acid. Attachment assays reveal that the RGD sequence is one
of the major cell attachment sites in entactin and that this sequence is recognized by the α
β3 integrin receptor. Analysis of cell attachment on mutant forms of full-length entactin expressed in the baculovirus expression
system revealed a second attachment site that was independent of the RGD sequence. This second site was localized to a peptide
of 39 amino acid residues in the second globular G2 domain of entactin. This peptide represents a cysteine-rich EGF repeat.
Inhibition of cell attachment by anti-integrin receptor antibodies indicates that the second attachment site is recognized
by a member of the β1 family of integrin receptors, possibly α3β1.
Footnotes
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↵* This work was supported by Grants CA-21246 and GM-25690 from the National Institutes of Health. 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.
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↵1 The abbreviations used are:
- EGF
-
epidermal growth factor
- MMT
-
mouse mammary tumor
- PBS
-
phosphate-buffered saline
- CHAPS
-
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid
- PAGE
-
polyacrylamide gel electrophoresis
- GST
-
glutathione S-transferase
- mAb
-
monoclonal antibody.
