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.
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 alpha v beta 3 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 beta 1 family of integrin receptors, possibly alpha 3 beta 1.
Dong
L J
LJ
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.
Hsieh
J C
JC
Chung
A E
AE
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002448:Cell Adhesion; D003545:Cysteine; D004815:Epidermal Growth Factor / Q000737:chemistry*; D006801:Humans; D016023:Integrins / Q000502:physiology; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry*
D016023:Integrins; D008562:Membrane Glycoproteins; D009842:Oligopeptides; C029967:nidogen; D004815:Epidermal Growth Factor; C047981:arginyl-glycyl-aspartic acid; D003545:Cysteine