A Model for Transmembrane Signalling by the Aspartate Receptor Based on Random-cassette Mutagenesis and Site-directed Disulphide Cross-linking

Abstract

Extracellular information is transduced by transmembrane receptors into the inside of the cell across a membrane barrier. To understand the molecular basis of transmembrane signalling, we replaced the transmembrane segment 2 (TM2) of the Escherichia coliaspartate receptor, Tar, with random sequences that are 21 amino acid residues in length and consist of Arg, Gly, Ser, Cys, Val, Leu, Ile and Phe at each position. From this ensemble for recombinant molecules, functional receptors were recovered as clones that could bind aspartate and transmit a signal to the intracellular domain.Restricted average hydrophobicity values were observed for functional transmembrane domains, and support the observation that transmembrane segments typically havehydrophobicity values greater than 1.6. However, non-functional transmembrane domains with greater hydrophobicity than 1.6 indicate that hydrophobicity is not a sole determinant for its function. Fourier transform analysis of the functional TM2 sequences suggest that the transmembrane segment has an α-helical structure with three distinct faces. Cross-linking of the faces to transmembrane segment 1 (TM1) mimics the “locked” signalling phenotypes of the wild-type receptor. The results are consistent with a model in which TM2 rotates in the plane of the lipid bilayer, and the rotation becomes locked at one face of the α-helix in the presence of attractant and at another face in the presence of repellent. This dynamic movement of the transmembrane domain may be a common signalling mechanism of homologous membrane receptor molecule such as the insulin receptor. Random-cassette mutagenesis and disulphde cross-linking provide powerful strategies for examining the structure and function of transmembrane segments.