Spinophilin stabilizes cell surface expression of alpha 2B-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) < or = 60 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 p75NTR extended the half-life of alpha 2B-AR at the apical surface to approximately 3.6 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 alpha2-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.
Brady
Ashley E
AE
Department of Pharmacology, Center for Molecular Neuroscience, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
Wang
Qin
Q
Colbran
Roger J
RJ
Allen
Patrick B
PB
Greengard
Paul
P
Limbird
Lee E
LE
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D005260:Female; D005455:Fluorescent Antibody Technique; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000502:physiology*; D011247:Pregnancy; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D000099273:Neurabins
D000316:Adrenergic alpha-Agonists; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D018341:Receptors, Adrenergic, alpha-2; D000099273:Neurabins