A role for PKD1 and PKD3 activation in modulation of calcium oscillations induced by orexin receptor 1 stimulation

The neuropeptides orexin-A/hypocretin-1 (Ox-A) and orexin-B/hypocretin-2 play an important role in the control of energy metabolism via either of two G-protein-coupled receptors, orexin receptor 1 (Ox1R) and 2. Despite its significant physiological functions, signaling via orexin receptors is still poorly characterized. The aim of this study was to improve our understanding of early signaling events triggered by the binding of Ox-A to Ox1R. Using phosphospecific antibodies, we observed that early kinase activation by Ox-A in a HEK293 cell line stably expressing Ox1R (HEKOx1R) included ERK1/2, PKCδ, and PKD1. Elevation of intracellular Ca2+ is a well-characterized response to Ox1R activation. Comparison of Ox-A-induced calcium elevation and PKD1 activation demonstrated that both responses are detectable soon after stimulation and increase in a dose-dependent manner, but inhibition of protein kinase C, when low Ox-A concentrations are used, affects them differently. PKD family of protein kinases has 3 members: PKD1, 2, and 3, which are all expressed in HEKOx1R cells. In response to stimulation of the cells with 1 nM Ox-A, both PKD1 and PKD3 are activated and increased in the plasma membrane, pointing at a possible role for these kinases in that cell compartment. Overexpression of either kinase-dead PKD1 or kinase-dead PKD3 disrupts Ox-A-induced calcium oscillations demonstrating the functional role of these kinases in modulating physiological responses to Ox-A.

Research Highlights
► Orexin-A/hypocretin-1 binding to orexin receptor 1 triggers a rapid intracellular calcium rise and activation of the protein kinases PKD1 and 3. This calcium response consists of oscillations which frequency and pattern are modulated by PKD1 and PKD3.