Identification of residues involved in homodimer formation located within a β-strand region of the N-terminus of a Yeast G protein-coupled receptor

J Recept Signal Transduct Res. 2012 Apr;32(2):65-75. doi: 10.3109/10799893.2011.647352. Epub 2012 Jan 24.

Abstract

G protein-coupled receptors (GPCRs) are members of a superfamily of cell surface signaling proteins that play critical roles in many physiological functions; malfunction of these proteins is associated with multiple diseases. Understanding the structure-function relationships of these proteins is important, therefore, for GPCR-based drug discovery. The yeast Saccharomyces cerevisiae tridecapeptide pheromone α-factor receptor Ste2p has been studied as a model to explore the structure-function relationships of this important class of cell surface receptors. Although transmembrane domains of GPCRs have been examined extensively, the extracellular N-terminus and loop regions have received less attention. We have used substituted cysteine accessibility method to probe the solvent accessibility of single cysteine residues engineered to replace residues Gly20 through Gly33 of the N-terminus of Ste2p. Unexpectedly, our analyses revealed that the residues Ser22, Ile24, Tyr26, and Ser28 in the N-terminus were solvent inaccessible, whereas all other residues of the targeted region were solvent accessible. The periodicity of accessibility from residues Ser22-Ser28 is indicative of an underlying structure consistent with a β-strand that was predicted computationally in this region. Moreover, a number of these Cys-substituted Ste2p receptors (G20C, S22C, I24C, Y26C, S28C and Y30C) were found to form increased dimers compared to the Cys-less Ste2p. Based on these data, we propose that part of the N-terminus of Ste2p is structured and that this structure forms a dimer interface for Ste2p molecules. Dimerization mediated by the N-terminus was affected by ligand binding, indicating an unanticipated conformational change in the N-terminus upon receptor activation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Substitution
  • Binding Sites
  • Cysteine / chemistry
  • Cysteine / genetics
  • Cysteine / metabolism*
  • Immunoblotting
  • Mutagenesis, Site-Directed
  • Protein Binding
  • Protein Multimerization*
  • Protein Structure, Secondary*
  • Receptors, Mating Factor / chemistry*
  • Receptors, Mating Factor / genetics
  • Receptors, Mating Factor / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Signal Transduction

Substances

  • Receptors, Mating Factor
  • STE2 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Cysteine