Liang Wei, Curran Patricia K, Hoang Quang, Moreland R Travis, Fishman Peter H
Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, National Institute of Neurological Disorders and Stroke, The National Institutes of Health, Bethesda, MD 20892, USA.
J Cell Sci. 2004 Feb 15;117(Pt 5):723-34. doi: 10.1242/jcs.00878. Epub 2004 Jan 20.
The beta(2)-adrenergic receptor (beta(2)AR) undergoes agonist-mediated endocytosis via clathrin-coated pits by a process dependent on both arrestins and dynamin. Internalization of some G protein-coupled receptors, however, is independent of arrestins and/or dynamin and through other membrane microdomains such as caveolae or lipid rafts. The human beta(1)AR is less susceptible to agonist-mediated internalization than the beta(2)-subtype, and its endocytic route, which is unknown, may be different. We have found that (i) co-expression of arrestin-2 or -3 enhanced the internalization of both subtypes whereas co-expression of dominant-negative mutants of arrestin-2 or dynamin impaired their internalization, as did inhibitors of clathrin-mediated endocytosis. (ii) Agonist stimulation increased the phosphorylation of beta(2)AR but not beta(1)AR. (iii) In response to agonist, each subtype redistributed from the cell surface to a distinct population of cytoplasmic vesicles; those containing beta(1)AR were smaller and closer to the plasma membrane whereas those containing beta(2)AR were larger and more perinuclear. (iv) When subcellular fractions from agonist-treated cells were separated by sucrose density gradient centrifugation, all of the internalized beta(2)AR appeared in the lighter endosomal-containing fractions whereas some of the internalized beta(1)AR remained in the denser plasma membrane-containing fractions. (v) Both subtypes recycled with similar kinetics back to the cell surface upon removal of agonist; however, recycling of beta(2)AR but not beta(1)AR was inhibited by monensin. Based on these results, we propose that the internalization of beta(1)AR is both arrestin- and dynamin-dependent and follows the same clathrin-mediated endocytic pathway as beta(2)AR. But during or after endocytosis, beta(1)AR and beta(2)AR are sorted into different endosomal compartments.
β₂ - 肾上腺素能受体（β₂AR）通过网格蛋白包被小窝进行激动剂介导的内吞作用，此过程依赖于抑制蛋白和发动蛋白。然而，一些G蛋白偶联受体的内化作用不依赖于抑制蛋白和/或发动蛋白，而是通过其他膜微区，如小窝或脂筏。人β₁AR比β₂亚型对激动剂介导的内化作用更不敏感，其未知的内吞途径可能有所不同。我们发现：（i）共表达抑制蛋白 - 2或 - 3增强了两种亚型的内化作用，而共表达抑制蛋白 - 2或发动蛋白的显性负性突变体则损害了它们的内化作用，网格蛋白介导的内吞作用抑制剂也有同样的效果。（ii）激动剂刺激增加了β₂AR的磷酸化，但未增加β₁AR的磷酸化。（iii）响应激动剂时，每种亚型从细胞表面重新分布到不同的细胞质囊泡群体中；含有β₁AR的囊泡较小且更靠近质膜，而含有β₂AR的囊泡较大且更多位于核周。（iv）当用蔗糖密度梯度离心法分离激动剂处理细胞的亚细胞组分时，所有内化的β₂AR都出现在较轻的含内体组分中，而一些内化的β₁AR仍留在较密的含质膜组分中。（v）去除激动剂后，两种亚型以相似的动力学循环回到细胞表面；然而，莫能菌素抑制了β₂AR的循环，但未抑制β₁AR的循环。基于这些结果，我们提出β₁AR的内化作用既依赖于抑制蛋白又依赖于发动蛋白，并且遵循与β₂AR相同的网格蛋白介导的内吞途径。但在内吞过程中或之后，β₁AR和β₂AR被分选到不同的内体区室中。
