Research Summary

Structural basis of ligand recognition by integrins

The capacity of cells to recognize other cells and specific elements of their surrounding extracellular matrix plays a central role in a diverse range of cellular processes including cellular differentiation, cell migration, the immune response and the maintenance of tissue architecture. These adhesive interactions are mediated by a limited number of cell surface adhesion receptor families. Unique among these adhesion receptor families is the Integrin receptor family whose members mediate both cell-cell and cell-matrix interactions. It is now recognized that integrins are not just simple adhesion receptors but in fact are complex bi-directional signaling molecules. Cellular activation by various agonists results in inside-out signaling allowing the integrins to rapidly modulate their affinity for ligands. Ligand binding results in outside-in signals with subsequent changes in cell behavior.

A major focus of the lab has been an investigation of structure-function relationships of the major platelet integrin alphaIIb-beta3 in order to provide a precise molecular map that defines the structural basis which underlies the interaction of adhesive ligands with platelets. Platelet aggregation and platelet adherence to components of the extracellular matrix in areas of vessel or tissue trauma are control events in hemostasis and thrombosis. The importance of these platelet adhesive interactions is underscored by the observations that abnormalities in these processes are often associated with pathologies that range from severe bleeding disorders to organ failure as a consequence of thrombotic occlusion of vessels. These interactions are mediated in large part by the binding of a set of adhesive proteins to the integrin alphaIIb-beta3. Elucidation of the mechanism for ligand recognition is central to the understanding of alphaIIb-beta3 receptor function. Moreover, determination of the molecular basis of ligand recognition by alphaIIb-beta3 is the first step towards the modulation of platelet function and thus may provide new insights into the development of antithrombotic strategies.

The Beta subunit plays a role in a common feature of ligand binding by integrins

We have sought to identify residues essential for receptor function by determining the mutations present in natural receptor variants characterized by the loss of receptor function despite normal expression levels. A single amino acid substitution (Asp119 to Tyr) in beta3 completely abolishes the ligand binding function of beta3 integrins. This residue is absolutely conserved among all integrin beta subunits and mutation of the corresponding residue in other integrin beta subunits similarly abrogates receptor function indicating a role for this residue in a common feature of ligand binding by integrins. A common feature of ligand binding to all integrins is an absolute dependence on divalent cations. In this regard, it is noteworthy that this variant alphaIIb-beta3 is associated with a perturbed interaction with divalent cations. Mutational analysis of residues proximal to Asp119 has assigned critical functional roles to two adjacent serine residues in the ligand binding function of alphaIIb-beta3. Together, these residues in alphaIIb-beta3 comprise a DxSxS sequence (in the single letter amino acid code where x is any residue) that is absolutely conserved in all the integrin beta subunits. This essential DxSxS motif is also highly conserved in the I (Inserted) domain present in six of the integrin alpha subunits. Utilizing the crystal structure of a recombinant I domain, we have modeled the homologous region in the beta3 subunit and have identified additional non-contiguous amino acids with oxygenated side chains essential for ligand binding function of the receptor. Substitution of these residues results in loss of ligand binding and perturbed interaction with divalent cation. These results further define a novel cation binding motif essential for integrin receptor function. Ongoing studies are utilizing recombinant beta3 fragments to obtain high resolution structures for this module and to map its interaction with cation and ligand.

Basis of ligand recognition specificity

The integrin alpha subunits play a major role in the regulation of ligand binding specificity. Within integrin subfamilies, a common alpha subunit can combine with different beta subunits to form receptors with distinct ligand recognition specificities. To gain further insight into the regions of the alpha subunits that regulate ligand specificity, we have utilized alphav/alphaIIb chimeras to identify regions of alphaIIb capable of switching the ligand binding phenotype of alphav-beta3 to that of alphaIIb-beta3. We have determined that the ligand recognition specificity of beta3 integrins is regulated by the amino terminal one-third of the alpha subunit. The identification of a number of potential ligand contact points implies that multiple sites are involved in ligand-receptor interaction. Effective ligand binding may require the sequential engagement of different sites. Studies are ongoing to identify discrete residues that define ligand binding specificity utilizing computer models of the structure of the NH2-terminal domain of integrin alpha subunits. Identification of specific ligand contact points may provide valuable insight into the development of agents to selectively modulate the function of the beta3 integrins and additional insights into wound healing and vascular proliferation.

Integrin function in cardiac myocytes

Integrin mediated cell-cell and cell-extracellular matrix interactions play a central role in the adhesive functions of vascular cells. Current methods, such as gene knockouts, to study integrin function in differentiated cells are not practical approaches since they have proven to be prohibitive of normal development. The identification of structural elements in integrins that abolish or enhance ligand binding function provides a unique, alternative means to alter the adhesive properties of cells in an integrin class specific and tissue specific manner. To explore this potential, we are examining the effects of expression of integrin mutants, which exhibit either loss of function or gain of function, in vascular cells in vitro and in transgenic mice.

We are using both loss of function and signaling mutants to assess the role of integrin adhesion and signaling in the hypertrophic response of cardiac myocytes. Increased mechanical loading leads to hypertrophic growth of the terminally differentiated cardiac myocyte. While this process is initially compensatory, the signaling pathways that lead from compensated hypertrophy to decompensated heart failure are presently unknown. The capacity of integrins to function as stretch receptors and mechanotransducers makes ideal candidates for translating abnormal strain into intracellular signals. We have been able to directly link integrin signaling to the hypertrophic response of cardiac myocytes in a cell culture model. Important pathways and critical effector molecules identified in these in vitro studies will be targeted in transgenic mice to obtain further insights into the role of integrins in cardiac function.