DESIGN AND CHEMICAL SYNTHESIS OF A NEOPROTEIN STRUCTURAL MODEL FOR THE CYTOPLASMIC DOMAIN OF A MULTISUBUNIT CELL-SURFACE RECEPTOR - INTEGRIN ALPHA(IIB)BETA(3) (PLATELET GPIIB-IIIA)

Integrins are a class of heterodimeric cell adhesion receptors involve d in cell migration, cell anchorage, and cell-cell interactions. The c ytoplasmic domains of integrins are of key importance in these activit ies. We have designed and chemically synthesized a 126 amino acid mode l protein (MP-1) containing both cytoplasmic tails of the platelet-der ived integrin alpha(IIb)beta(3) covalently linked via a helical coiled coil. The coiled-coil tertiary structure was incorporated to mimic th e membrane-spanning domain of the integrin and to act as a topological constraint fixing the two cytoplasmic tails in a parallel arrangement . This molecule, which contains two C-termini, was constructed by chem ical dovetailing. The bromoacetylated and cysteinyl peptide synthons w ere unambiguously ligated through the formation of a thioether linkage . Ultraviolet circular dichroism (CD) spectroscopy has been performed on MP-1 and related compounds, confirming that a helical coiled coil i s present within the MP-1 molecule. Significantly, the helicity appare ntly extends beyond the predicted amphiphilic region of MP-1. Fluoresc ence measurements suggest that a defined tertiary structure has formed by the association of the two cytoplasmic domains. We conclude that t his is a practical design strategy for the study of the cytoplasmic do main of multisubunit cell-surface receptors.Integrins are a class of h eterodimeric cell adhesion receptors involved in cell migration, cell anchorage, and cell-cell interactions. The cytoplasmic domains of inte grins are of key importance in these activities. We have designed and chemically synthesized a 126 amino acid model protein (MP-1) containin g both cytoplasmic tails of the platelet-derived integrin alpha(IIb)be ta(3) covalently linked via a helical coiled coil. The coiled-coil ter tiary structure was incorporated to mimic the membrane-spanning domain of the integrin and to act as a topological constraint fixing the two cytoplasmic tails in a parallel arrangement. This molecule, which con tains two C-termini, was constructed by chemical dovetailing. The brom oacetylated and cysteinyl peptide synthons were unambiguously ligated through the formation of a thioether linkage. Ultraviolet circular dic hroism (CD) spectroscopy has been performed on MP-1 and related compou nds, confirming that a helical coiled coil is present within the MP-1 molecule. Significantly, the helicity apparently extends beyond the pr edicted amphiphilic region of MP-1. Fluorescence measurements suggest that a defined tertiary structure has formed by the association of the two cytoplasmic domains. We conclude that this is a practical design strategy for the study of the cytoplasmic domain of multisubunit cell- surface receptors.