Received for publication April 13, 2006.
Revised August 22, 2006.
Accepted for publication August 22, 2006.

PROBING THE EXISTENCE OF G PROTEIN-COUPLED RECEPTOR DIMERS BY POSITIVE AND NEGATIVE LIGAND-DEPENDENT COOPERATIVE BINDING

Abstract

An increasing amount of ligand binding data on G protein-coupled receptors (GPCRs) is not compatible with the prediction of the simple mass action law. This may be related to the propensity of most GPCRs, if not all, to oligomerize. Indeed, one of the consequences of receptor oligomerisation could be a possible crosstalk between the protomers which in term could lead to negative or positive cooperative ligand binding. We prove here that this can be demonstrated experimentally. Saturation, dissociation and competition binding experiments were performed onto vasopressin and oxytocin receptors expressed in CHO or Cos-7 cells. Linear, concave and convex scatchard plots were then observed depending on the ligand used. Moreover, some competition curves exhibited an increase of the radiotracer binding for low concentrations of competitors suggesting a cooperative binding process. These data demonstrate that various vasopressin analogs display either positive or negative cooperative binding. Because positive cooperative binding cannot be explained without considering receptor as multivalent, these binding data support the concept of GPCR dimerization process. The results which are in good accordance with the predictions of previous mathematical models, suggest that binding experiments can be used to probe the existence of receptor dimers.

Key words: Neuropeptides, Vasopressin/Oxytocin, Structure-activity relationships and modeling, Fluorescence techniques, Peptide hormones

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