NCAM180 regulates Ric8A membrane localization and potentiates β-adrenergic response.

Cooperation between receptors allows integrated intracellular signaling leading to appropriate physiological responses. The Neural Cell Adhesion Molecule (NCAM) has three main isoforms of 120, 140 and 180 kDa, with adhesive and signaling properties, but their respective functions remains to be fully identified. Here we show that the human NCAM180 intracellular domain is a novel interactor of the human guanosine exchange factor (GEF) Ric8A using the yeast two hybrid system and immunoprecipitation. Furthermore, NCAM, Ric8A and G(αs) form a tripartite complex. Colocalization experiments by confocal microscopy revealed that human NCAM180 specifically induces the recruitment of Ric8A to the membrane. In addition, using an in vitro recombinant system, and in vivo by comparing NCAM knock-out mouse brain to NCAM heterozygous and wild type brains, we show that NCAM expression dose dependently regulates Ric8A redistribution in detergent resistent membrane microdomains (DRM). Previous studies have demonstrated essential roles for Ric8 in G(α) protein activity at G protein coupled receptors (GPCR), during neurotransmitter release and for asymmetric cell division. We observed that inhibition of Ric8A by siRNA or its overexpression, decreases or increases respectively, cAMP production following β-adrenergic receptor stimulation. Furthermore, in human HEK293T recombinant cells, NCAM180 potentiates the G(αs) coupled β-adrenergic receptor response, in a Ric8A dependent manner, whereas NCAM120 or NCAM140 do not. Finally, in mouse hippocampal neurons expressing endogenously NCAM, NCAM is required for the agonist isoproterenol to induce cAMP production, and this requirement depends on Ric8A. These data illustrate a functional crosstalk between a GPCR and an IgCAM in the nervous system.