Wang Qin, Limbird Lee E
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
J Biol Chem. 2002 Dec 27;277(52):50589-96. doi: 10.1074/jbc.M208503200. Epub 2002 Oct 9.
The present studies demonstrate that no single stretch of sequence in the third intracellular (3i) loop of the alpha(2A) adrenergic receptor (alpha(2A)-AR) can fully account for its previously described interactions with spinophilin (Richman, J. G., Brady, A. E., Wang, Q., Hensel, J. L., Colbran, R. J., and Limbird, L. E. (2001) J. Biol. Chem. 276, 15003-15008), 14-3-3zeta (Prezeau, L., Richman, J. G., Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 13462-13469), and arrestin 3 (Wu, G., Krupnick, J. G., Benovic, J. L., and Lanier, S. M. (1997) J. Biol. Chem. 272, 17836-17842), suggesting that a three-dimensional surface, rather than a linear sequence, provides the basis for these interactions as proposed for 3i loop tethering of the alpha(2A)-AR to the basolateral surface of Madin-Darby canine kidney cells (Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 16331-16336). Sequences at the extreme N-terminal and C-terminal ends of the 3i loop are critical for interaction with spinophilin but not for interaction with 14-3-3zeta or arrestin 3, for which the C-terminal half of the loop is more important. Competition binding for (35)S-labeled alpha(2A)-AR 3i loop binding to glutathione S-transferase (GST)-spinophilin amino acids 151-444 revealed a relative affinity of spinophilin congruent with arrestin > 14-3-3zeta for the unphosphorylated alpha(2A)-AR 3i loop. Agonist occupancy of the alpha(2A)-AR increases receptor association with spinophilin, and arrestin 3 appears to compete for this enrichment. However, when the G protein-coupled receptor kinase 2 substrate sequence was deleted from the 3i loop, arrestin 3 could not compete for the agonist-enriched binding of spinophilin to the mutant alpha(2A)-AR. These data are consistent with a model where sequential or competitive interactions among spinophilin, arrestin, and/or 14-3-3zeta play a role in alpha(2A)-AR functions.
目前的研究表明，α₂A肾上腺素能受体（α₂A-AR）第三细胞内环（3i环）中没有单一的连续序列能够完全解释其先前所述的与亲嗜素（Richman, J. G., Brady, A. E., Wang, Q., Hensel, J. L., Colbran, R. J., and Limbird, L. E. (2001) J. Biol. Chem. 276, 15003 - 15008）、14-3-3ζ（Prezeau, L., Richman, J. G., Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 13462 - 13469）和抑制蛋白3（Wu, G., Krupnick, J. G., Benovic, J. L., and Lanier, S. M. (1997) J. Biol. Chem. 272, 17836 - 17842）的相互作用，这表明三维表面而非线性序列为这些相互作用提供了基础，正如α₂A-AR通过3i环拴系于Madin-Darby犬肾细胞基底外侧表面所提出的那样（Edwards, S. W., and Limbird, L. E. (1999) J. Biol. Chem. 274, 16331 - 16336）。3i环极端N末端和C末端的序列对于与亲嗜素的相互作用至关重要，但对于与14-3-3ζ或抑制蛋白3的相互作用并非如此，对于后者，环的C末端一半更为重要。针对（³⁵）S标记的α₂A-AR 3i环与谷胱甘肽S-转移酶（GST）-亲嗜素氨基酸151 - 444的竞争结合显示，对于未磷酸化的α₂A-AR 3i环，亲嗜素的相对亲和力与抑制蛋白＞14-3-3ζ相当。α₂A-AR的激动剂占据增加了受体与亲嗜素的结合，并且抑制蛋白3似乎竞争这种富集。然而，当G蛋白偶联受体激酶2底物序列从3i环中缺失时，抑制蛋白3无法竞争亲嗜素与突变型α₂A-AR的激动剂富集结合。这些数据与一个模型一致，即亲嗜素、抑制蛋白和/或14-3-3ζ之间的顺序或竞争相互作用在α₂A-AR功能中起作用。
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