Spinophilin Stabilizes Cell Surface Expression of alpha sub(2B)-Adrenergic Receptors

• Published in: The Journal of biological chemistry Vol. 278; no. 34; pp. 32405 - 32412
• Main Authors: Brady, A E, Wang, Q, Colbran, R J, Allen, P B, Greengard, P, Limbird, LE
• Format: Journal Article
• Language: English
• Published: 22.08.2003
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M304195200

The third intracellular (3i) loops of the alpha sub(2A)- and alpha sub(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 sub(2A), alpha sub(2B), and alpha sub(2C)-AR subtypes interact with spinophilin, a multidomain protein that, like the three alpha sub(2)-AR subtypes, is enriched at the basolateral surface of MDCKII cells. The present studies provide evidence that alpha sub(2)-AR interaction with spinophilin contributes to cell surface stabilization of the receptor. We exploited the unique targeting profile of the alpha sub(2B)-AR subtype in MDCKII cells: random delivery to apical and basolateral surfaces with rapid (t sub([1/2]) <= 60 min) apical versus slower (t sub([1/2]) = 10-12 h) basolateral turnover. Apical delivery of a spinophilin subdomain containing the alpha sub(2)-AR-interacting region (Sp151-483) by fusion with apically targeted p75 super(NTR) extended the half-life of alpha sub(2B)-AR at the apical surface to similar to 3.6 h and eliminated the rapid phase (0-60 min) of alpha sub(2B)-AR turnover on that surface. Furthermore, we examined alpha sub(2B)-AR turnover at the surface of mouse embryo fibroblasts derived from wild type (Sp super(+) super(/) super(+)) or spinophilin knock-out (Sp super(-) super(/) super(-)) mice. Two independent experimental approaches demonstrated that agonist-evoked internalization of HA- alpha sub(2B)-AR was accelerated in mouse embryo fibroblasts derived from Sp super(-) super(/) super(-) mice. These findings are consistent with the interpretation that endogenous spinophilin contributes to the stabilization of alpha sub(2B)-AR and presumably all three alpha sub(2)-AR subtypes at the surface of target cells and may act as a scaffold that could link alpha sub(2)-ARs to proteins interacting with spinophilin via other domains.