G-Protein coupling and signalling of Y1-like neuropeptide Y receptors in SK-N-MC cells

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Abstract
Summary We have studied [125I]neuropeptide Y-binding sites and neuropeptide Y-mediated second messenger responses in human SK-N-MC neuroblastoma cells with special reference to the role of G-proteins. Neuropeptide Y stimulated two second messenger responses in SK-N-MC cells, inhibition of cAMP accumulation and mobilization of Ca2+ from intracellular stores. Both effects were completely abolished by pretreatment with pertussis toxin. Binding of [125I]neuropeptide Y to intact cells or SK-N-MC cell membranes was rapid, reversible, characterized by high affinity and low capacity, and had pharmacological characteristics of a homogeneous population of Y1-like neuropeptide Y receptors. In permeabilized cells, [125I] neuropeptide Y binding was inhibited by GTPγ S in a concentration-dependent manner. Saturation experiments in the absence and presence of GTPγS demonstrated a reduction in the number of high-affinity [125I]neuropeptide Y-binding sites without a decrease in affinity of the remaining sites. Pretreatment of intact cells with ertussis toxin completely abolished the inhibition of [125I]neuropeptide Y binding by GTPγS. Moreover, pertussis toxin treatment reduced the number of high-affinity [125I]neuropeptide Y binding sites. We conclude that the agonist ligand [125I]neuropeptide Y identifies functional neuropeptide Y receptors in SK-N-MC cells; however, the number of specific [125I]neuropeptide Y-binding sites may not necessarily reflect the number of neuropeptide Y receptors, because the former is affected by the functional state of cellular G-proteins.