title: A model for transmembrane signalling by the aspartate receptor based on random-cassette mutagenesis and site-directed disulfide 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 coli aspartate 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 have hydrophobicity 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 suggests that the transmembrane segment has an alpha-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 alpha-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 molecules such as the insulin receptor. Random-cassette mutagenesis and disulfide cross-linking provide powerful strategies for examining the structure and function of transmembrane segments.
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000502:physiology; D002633:Chemotaxis; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017459:Receptors, Amino Acid / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D003432:Cross-Linking Reagents; D004220:Disulfides; D017459:Receptors, Amino Acid; C031959:aspartic acid receptor; D003545:Cysteine