Isoproterenol-induced subcellular redistribution of G-protein β subunits in S49 lymphoma cells demonstrated by a novel competitive ELISA

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Abstract
A novel competitive ELISA has been developed/or the determination of levels of the β subunit ofguanine-nucleotide-binding protein (G-protein) using antipeptide antibodies directed against the amino terminus of the β subunit. Because β subunits form highly hydrophobic heterodimeric complexes with γ subunits of G-proteins, specific assay conditions were required. Optimal concentrations of antibodies, detergents, Mg2+ as well as ionic strength were determined. In addition, we found that an effective binding of the used antibodies to the β subunit was ensured only after denaturation of the βγ complexes. Subsequently, this ELISA was used for quantitation of the β subunit in subcellular fractions of S49 lymphoma cells during isoproterenol-mediated desensitization of β-adrenergic controlled transmembrane signalling system. A 10 min as well as 60 min treatment of the cells with isoproterenol (1 nmol/ml) resulted in a significant shift of G-protein β subunits (presumably as βγ complexes) from the plasma membrane fractions to low-density microsomal fractions. No significant change was detected after the hormone action in the distribution of plasma membrane constitutive enzymes. In conclusion, the developed ELISA helped us to reveal that β-adrenergic stimulation can induce redistribution of the βγ dimer from plasma membranes to low-density microsomes.