Spinophilin stabilizes cell surface expression of α2B-adrenergic receptors

The third intracellular (3i) loops of the alpha(2A)- and alpha(2B)-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MDCKII) cells at steady state. The third intracellular loops of the alpha(2A), alpha(2B), and alpha(2C)-AR subtypes interact with spinophilin, a multidomain protein that, like the three alpha(2)-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that alpha(2)-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the alpha(2B)-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t(1/2)less than or equal to60 min) apical versus slower (t(1/2)=10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the alpha(2)-AR-interacting region (Sp151-483) by fusion with apically targeted p75(NTR) extended the half-life of alpha(2B)-AR at the apical surface to similar to3.6 h and eliminated the rapid phase (0-60 min) of alpha(2B)-AR turnover on that surface. Furthermore, we examined alpha(2B)-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp(+/+)) or spinophilin knock-out (Sp(-/-)) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA-alpha(2B)-AR was accelerated in mouse embryo fibroblasts derived from Sp(-/-) mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of alpha(2B)-AR and presumably all three alpha(2)-AR subtypes at the surface of target cells and may act as a scaffold that could link alpha(2)-ARs to proteins interacting with spinophilin via other domains.