Received for publication July 29, 2004.
Revised February 10, 2005.
Accepted for publication February 10, 2005.

Alpha2-adrenergic agonist enrichment of spinophilin at the cell surface involves subunits of G_i proteins and is preferentially induced by the _2A-subtype

Abstract

Agonist activation regulates reciprocal interactions of spinophilin and arrestin with the _2A- and _2B-adrenergic receptor (AR) subtypes via their 3i loop. Since arrestin association with GPCR is preceded by redistribution of arrestin to the cell surface, the present studies explored whether agonist activation of the _2A- and _2B-AR subtypes also led to spinophilin enrichment at the cell surface. Live cell imaging studies using a green-fluorescent protein (GFP)-tagged spinophilin examined spinophilin localization, and its regulation by ₂-AR agonist. Agonist activation of _2A-AR preferentially, when compared to _2B-AR, led to spinophilin enrichment at the cell surface in HEK293 cells and in mouse embryo fibroblasts derived from spinophilin null mice. Activation of the LEESSSS_2A-AR, which has enriched association with spinophilin compared to the WT _2A-AR, does not show an enhanced redistribution of spinophilin to the surface when compared to WT _2A-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 appears to involve downstream signaling events, manifest both by the pertussis toxin sensitivity of the process and by the marked attenuation of spinophilin redistribution in cells expressing the ARK-C tail, which sequesters subunits of G proteins. Taken together, the data suggest that agonist-evoked spinophilin enrichment at the cell surface is due to receptor-evoked signaling pathways and is independent of the affinity of the receptor for the spinophilin molecule.

Key words: Adrenergic, Gi family, Fluorescence techniques

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