Planta

, Volume 221, Issue 5, pp 667–674

LeRALF, a plant peptide that regulates root growth and development, specifically binds to 25 and 120 kDa cell surface membrane proteins of Lycopersicon peruvianum

  • Justin M. Scheer
  • Gregory Pearce
  • Clarence A. Ryan
Original Article

Abstract

A photoaffinity analog of tomato leaf RALF peptide (LeRALF), 125I-azido-LeRALF, bound saturably to tomato suspension cultured cells in the dark in a classical receptor binding assay. Classical kinetic analyses revealed that the analog interacted with a single binding site on the surface of the cells with a KD of 0.8×10−9 M, typical of known peptide hormone–receptor interactions in both plants and animals. The 125I-azido-LeRALF, when exposed to UVB light in the presence of the cells, strongly labeled only two proteins of 25 kDa and 120 kDa, with the 25 kDa protein being more strongly labeled than the 120 kDa protein. The cell-surface localization of the interaction was indicated, as suramin, a known inhibitor of peptide–receptor interactions, and native LeRALF peptide competed with 125I-azido-LeRALF labeling of both proteins. Two biologically inactive LeRALF analogs were not competitors. Incubation of 125I-azido-LeRALF with suspension cultured cells in the dark, where it was fully active, could subsequently be totally dissociated from cells by acid washes, indicating that it was interacting at the cell surface and was not internalized. The 125I-azido-LeRALF-labeled 25 kDa and 120 kDa proteins could not be solubilized from cell membranes by methods that release peripheral proteins, indicating that they are integral membrane components. The cumulative kinetic and biochemical evidence strongly indicates that the two proteins may be components of a LeRALF receptor complex.

Keywords

Alkalinization assay Peptide signaling Photoaffinity labeling Receptor Suramin Suspension cultured cells 

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Justin M. Scheer
    • 1
    • 2
  • Gregory Pearce
    • 1
  • Clarence A. Ryan
    • 1
  1. 1.Institute of Biological ChemistryWashington State UniversityPullmanUSA
  2. 2.Department of BiologySunesis Pharmaceuticals, IncSouth San FranciscoUSA

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