Spinophilin stabilizes cell surface expression of α _2B-adrenergic receptors

The third intracellular (3i) loops of the α_2A- and α_2B-adrenergic receptor (AR) subtypes are critical for retention of these receptors at the basolateral surface of polarized Madin-Darby canine kidney (MIICKII) cells at steady state. The third intracellular loops of the α_2A, α_2B, and α_2C-AR subtypes interact with spinophilin, a multidomain protein that, like the three α₂-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that α₂-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the α_2B-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t_1/2 ≤ 60 min) apical versus slower (t_1/2 = 10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the α ₂-AR-interacting region (Sp151-483) by fusion with apically targeted p75^NTR extended the half-life of α_2B-AR at the apical surface to ∼3.6 h and eliminated the rapid phase (0-60 min) of α_2B-AR turn-over on that surface. Furthermore, we examined α_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-α_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 β_2B-AR and presumably all three α₂-AR subtypes at the surface of target cells and may act as a scaffold that could link α₂-ARs to proteins interacting with spinophilin via other domains.