title: Alpha 2-adrenergic agonist enrichment of spinophilin at the cell surface involves beta gamma subunits of Gi proteins and is preferentially induced by the alpha 2A-subtype.
abstract: Agonist activation regulates reciprocal interactions of spinophilin and arrestin with the alpha2A- and alpha2B -adrenergic receptor (AR) subtypes via their 3i loop. Because arrestin association with G protein-coupled receptor is preceded by redistribution of arrestin to the cell surface, the present studies explored whether agonist activation of the alpha2A- and alpha2B -AR subtypes also led to spinophilin enrichment at the cell surface. Live cell imaging studies using a green fluorescent protein-tagged spinophilin examined spinophilin localization and its regulation by alpha2 -AR agonist. Agonist activation of alpha2A-AR preferentially, compared with the alpha2B-AR, led to spinophilin enrichment at the cell surface in human embryonic kidney 293 cells and in mouse embryo fibroblasts derived from spinophilin null mice. Activation of the delta LEESSSS alpha2A-AR, which has enriched association with spinophilin compared with the wild-type (WT) alpha2A-AR, does not show an enhanced redistribution of spinophilin to the surface compared with WT alpha2A-AR, demonstrating that the ability or affinity of the receptor in binding spinophilin may be independent of the ability of the receptor to effect spinophilin redistribution to the surface. Agonist-evoked enrichment of spinophilin at the cell surface seems to involve downstream signaling events, manifested both by the pertussis toxin sensitivity of the process and by the marked attenuation of spinophilin redistribution in cells expressing the beta-adrenergic receptor kinase-C tail, which sequesters beta gamma subunits of G proteins. Together, the data suggest that agonist-evoked spinophilin enrichment at the cell surface is caused by receptor-evoked signaling pathways and is independent of the affinity of the receptor for the spinophilin molecule.
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
authors.forename: Ashley E
authors.initials: AE
authors.lastname: Brady
authors.forename: Qin
authors.initials: Q
authors.lastname: Wang
authors.forename: Patrick B
authors.initials: PB
authors.lastname: Allen
authors.forename: Mark
authors.initials: M
authors.lastname: Rizzo
authors.forename: Paul
authors.initials: P
authors.lastname: Greengard
authors.forename: Lee E
authors.initials: LE
authors.lastname: Limbird
mesh_terms: D058647:Adrenergic alpha-2 Receptor Agonists; D000316:Adrenergic alpha-Agonists / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism; D000099273:Neurabins
chemical_list: D058647:Adrenergic alpha-2 Receptor Agonists; D000316:Adrenergic alpha-Agonists; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D018341:Receptors, Adrenergic, alpha-2; D000099273:Neurabins; C547768:ADRA2A protein, human; C547769:Adra2a protein, mouse