Gawaz M, Besta F, Ylänne J, Knorr T, Dierks H, Böhm T, Kolanus W
Laboratorium für Molekulare Biologie, Genzentrum der Universität München, Feodor Lynen Strasse 25, D-81377 München, Germany.
J Cell Sci. 2001 Mar;114(Pt 6):1101-13. doi: 10.1242/jcs.114.6.1101.
Beta3 integrin adhesion molecules play important roles in wound repair and the regulation of vascular development and three beta3 integrin isoforms (beta3-A, -B, -C) have been described so far. Surface expression of beta3 integrins is dynamically regulated through internalization of beta3 integrins, however, the molecular mechanisms are understood incompletely. To evaluate the role of the cytoplasmic domain of beta3 integrins for internalization, we have generated single chain chimeras with variant and mutated forms of beta3 cytoplasmic domains. Upon transient transfection into chinese hamster ovary cells, it was found that the beta3-A chimera had strongly reduced cell surface expression compared with the corresponding beta3-B, or beta3-C fusion proteins, or the tail-less constructs, whereas steady state levels of all chimeras were near identical. Studies employing cytoplasmic domain mutants showed that the NITY motif at beta3-A 756-759 is critical for plasma membrane expression of beta3-A. Furthermore, delivery of beta3-A to the cell surface was specifically modulated by the cytoplasmic protein beta3-endonexin, a previously described intracellular protein. Coexpression of the native, long form of beta3-endonexin, which does not interact with the beta3 tail, acted as a dominant negative inhibitor of beta3-A-internalization and enhanced steady-state surface expression of the beta3-A-chimera. Furthermore, anti-beta3 antibody-induced internalization of the native beta3 integrin (alpha(IIb)beta3 was dramatically reduced for the Tyr(759)-Ala substitution mutant (alpha(IIb)beta3) (Y759A) and expression of the long isoform of beta3-endonexin substantially decreased the internalization of wild-type alpha(IIb)beta3. Thus, the NITY motif of the beta-chain cytoplasmic domain is involved in stimulated internalization of the beta3 integrin A isoform and beta3-endonexin appears to couple the beta3-A isoform to a specific receptor-recycling pathway.
β3整合素黏附分子在伤口修复以及血管发育调控中发挥着重要作用，迄今为止已发现三种β3整合素亚型（β3-A、-B、-C）。β3整合素的表面表达通过其内化作用受到动态调控，然而，其分子机制尚未完全明确。为评估β3整合素胞质结构域在内化过程中的作用，我们构建了具有β3胞质结构域变体和突变形式的单链嵌合体。将其瞬时转染至中国仓鼠卵巢细胞后发现，与相应的β3-B或β3-C融合蛋白或无尾构建体相比，β3-A嵌合体的细胞表面表达显著降低，而所有嵌合体的稳态水平几乎相同。利用胞质结构域突变体进行的研究表明，β3-A 756-759位的NITY基序对β3-A的质膜表达至关重要。此外，胞质蛋白β3-内毒素（一种先前描述的细胞内蛋白）特异性调节β3-A向细胞表面的转运。不与β3尾部相互作用的天然长形式β3-内毒素的共表达，作为β3-A内化的显性负抑制剂，增强了β3-A嵌合体的稳态表面表达。此外，对于Tyr(759)-Ala替代突变体（α(IIb)β3）（Y759A）而言，抗β3抗体诱导的天然β3整合素（α(IIb)β3）内化显著减少，并且β3-内毒素长亚型 的表达大幅降低了野生型α(IIb)β3的内化。因此，β链胞质结构域的NITY基序参与β3整合素A亚型的刺激内化过程，并且β3-内毒素似乎将β3-A亚型与特定的受体循环途径相偶联。
