The long term objective of this proposal is to derive an understanding of the protein biochemistry of cell adhesion to the extracellular matrix. This will be accomplished by examining structure-function relationships of two closely related cell adhesion receptors, integrins alphaIIBbeta3 and alphavbeta3. The proposed studies relate directly to the pathological biochemistry of cancer, osteoporosis and thrombosis. One of the key functional domains of the integrin adhesion receptors is the ligand binding site. Current opinion suggests that this domain encompasses amino-terminal regions of both the alpha and beta subunits of the integrins. However, a structural basis for the remarkably broad range of ligands that the integrins bind has not been put forth. the first specific aim of this proposal is to identify the region of the alpha subunit that confers ligand selection by integrins. This will be achieved by genetically engineering and expressing recombinant """"""""hybrid"""""""" receptors in an attempt to define the region of the receptor that regulates ligand binding and selection. This analysis will focus on substituting sequences from the alphaIIB subunit for homologous sequences within the alphav subunit. The resulting hybrid will be expressed with the beta3 subunit, and the ligand binding phenotype of the hybrid receptor will be assessed to determine which changes enact a phenotypic switch in ligand recognition. The integrin adhesion receptors also contain divalent cation binding sites which regulate the receptors activation state and ligand binding specificity. Little information is available regarding a mechanism and structural basis for this critical regulatory element.
The second aim of this proposal is to determine the number od divalent cation binding sites on the integrin which must be occupied for ligand binding to occur. This will be accomplished by performing direct binding studies between purified integrins and divalent cation. The central hypothesis of the proposed studies is that the site on the integrin alpha subunit that confers ligand binding specificity is identical to the divalent cation binding site that triggers ligand binding. this could explain the profound effect divalent cations have on ligand selection by the integrins. This hypothesis will be tested by identifying the location of the critical divalent cation binding site with a series of standard biochemical approaches including ligand blotting and site-specific protein modification. Results from this analysis will be compared to results from aim #1 to determine if the ligand contact site is also a regulatory divalent cation binding site. It is anticipated that successful completion of these aims will significantly advance current understanding of cell-extracellular matrix interactions.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA056483-04
Application #
2097345
Study Section
Pathobiochemistry Study Section (PBC)
Project Start
1993-02-01
Project End
1998-01-31
Budget Start
1995-06-01
Budget End
1996-01-31
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Sanford-Burnham Medical Research Institute
Department
Type
DUNS #
009214214
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Wong, N C; Mueller, B M; Barbas, C F et al. (1998) Alphav integrins mediate adhesion and migration of breast carcinoma cell lines. Clin Exp Metastasis 16:50-61
Smith, J W (1997) Allostery and proteolysis: two novel modes of regulating integrin function. Matrix Biol 16:173-8
Lin, E C; Ratnikov, B I; Tsai, P M et al. (1997) Evidence that the integrin beta3 and beta5 subunits contain a metal ion-dependent adhesion site-like motif but lack an I domain. J Biol Chem 272:14236-43
Lin, E C; Ratnikov, B I; Tsai, P M et al. (1997) Identification of a region in the integrin beta3 subunit that confers ligand binding specificity. J Biol Chem 272:23912-20
Seiffert, D; Smith, J W (1997) The cell adhesion domain in plasma vitronectin is cryptic. J Biol Chem 272:13705-10
Jongewaard, I N; Tsai, P M; Smith, J W (1996) The type III connecting segment of fibronectin contains an aspartic acid residue that regulates the rate of binding to integrin alpha 4 beta 1. Cell Adhes Commun 3:487-95
Hu, D D; Barbas, C F; Smith, J W (1996) An allosteric Ca2+ binding site on the beta3-integrins that regulates the dissociation rate for RGD ligands. J Biol Chem 271:21745-51
Stuiver, I; Ruggeri, Z; Smith, J W (1996) Divalent cations regulate the organization of integrins alpha v beta 3 and alpha v beta 5 on the cell surface. J Cell Physiol 168:521-31
Hu, D D; Hoyer, J R; Smith, J W (1995) Ca2+ suppresses cell adhesion to osteopontin by attenuating binding affinity for integrin alpha v beta 3. J Biol Chem 270:9917-25
Smith, J W; Tachias, K; Madison, E L (1995) Protein loop grafting to construct a variant of tissue-type plasminogen activator that binds platelet integrin alpha IIb beta 3. J Biol Chem 270:30486-90

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