Fluorescent muscarinic EGFP-hM1 chimeric receptors: design, ligand binding and functional properties

J Recept Signal Transduct Res. 1999 Jan-Jul;19(1-4):423-36. doi: 10.3109/10799899909036662.

Abstract

We describe the construction, expression and characterization of recombinant proteins comprising the enhanced green fluorescent protein (EGFP) fused to the amino-terminal part of the muscarinic hM1 receptor together or not with an additional hexahistidine tag placed at the C-terminal end of the receptor. Expression of the fluorescent proteins reaches levels identical to those of the wt hM1 receptor, provided that fusion takes place at the very N-terminal end of the receptor. Also correct protein folding and targeting to plasma membrane is obtained upon addition of a signal peptide promoting amino-terminal domain translocation through the membrane. Ligand binding properties of--and activation of the calcium release response by--the fusion proteins are almost identical to those of the wild-type muscarinic receptor, indicating that such fluorescently-labelled receptors are valuable model systems for further functional, biochemical and structural studies.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Line
  • DNA Primers / genetics
  • DNA, Complementary / genetics
  • Gene Expression
  • Green Fluorescent Proteins
  • Humans
  • Ligands
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Quinuclidinyl Benzilate / metabolism
  • Radioligand Assay
  • Receptor, Muscarinic M1
  • Receptors, Muscarinic / chemistry
  • Receptors, Muscarinic / genetics
  • Receptors, Muscarinic / metabolism*
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Spectrometry, Fluorescence
  • Transfection
  • Tritium

Substances

  • DNA Primers
  • DNA, Complementary
  • Ligands
  • Luminescent Proteins
  • Receptor, Muscarinic M1
  • Receptors, Muscarinic
  • Recombinant Fusion Proteins
  • Tritium
  • Green Fluorescent Proteins
  • Quinuclidinyl Benzilate