Pierce K L, Tohgo A, Ahn S, Field M E, Luttrell L M, Lefkowitz R J
Howard Hughes Medical Institute and the Departments of Medicine and Biochemistry, Box 3821, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2001 Jun 22;276(25):23155-60. doi: 10.1074/jbc.M101303200. Epub 2001 Apr 4.
"Transactivation" of epidermal growth factor receptors (EGFRs) in response to activation of many G protein-coupled receptors (GPCRs) involves autocrine/paracrine shedding of heparin-binding EGF (HB-EGF). HB-EGF shedding involves proteolytic cleavage of a membrane-anchored precursor by incompletely characterized matrix metalloproteases. In COS-7 cells, alpha(2A)-adrenergic receptors (ARs) stimulate ERK phosphorylation via two distinct pathways, a transactivation pathway that involves the release of HB-EGF and the EGFR and an alternate pathway that is independent of both HB-EGF and the EGFR. We have developed a mixed culture system to study the mechanism of GPCR-mediated HB-EGF shedding in COS-7 cells. In this system, alpha(2A)AR expressing "donor" cells are co-cultured with "acceptor" cells lacking the alpha(2A)AR. Each population expresses a uniquely epitope-tagged ERK2 protein, allowing the selective measurement of ERK activation in the donor and acceptor cells. Stimulation with the alpha(2)AR selective agonist UK14304 rapidly increases ERK2 phosphorylation in both the donor and the acceptor cells. The acceptor cell response is sensitive to inhibitors of both the EGFR and HB-EGF, indicating that it results from the release of HB-EGF from the alpha(2A)AR-expressing donor cells. Experiments with various chemical inhibitors and dominant inhibitory mutants demonstrate that EGFR-dependent activation of the ERK cascade after alpha(2A)AR stimulation requires Gbetagamma subunits upstream and dynamin-dependent endocytosis downstream of HB-EGF shedding and EGFR activation, whereas Src kinase activity is required both for the release of HB-EGF and for HB-EGF-mediated ERK2 phosphorylation.
表皮生长因子受体（EGFRs）对多种G蛋白偶联受体（GPCRs）激活的“反式激活”涉及肝素结合表皮生长因子（HB-EGF）的自分泌/旁分泌性脱落。HB-EGF的脱落涉及由特征不完全清楚的基质金属蛋白酶对膜锚定前体进行蛋白水解切割。在COS-7细胞中，α2A-肾上腺素能受体（ARs）通过两条不同的途径刺激ERK磷酸化，一条反式激活途径涉及HB-EGF和EGFR的释放，另一条替代途径独立于HB-EGF和EGFR。我们开发了一种混合培养系统来研究COS-7细胞中GPCR介导的HB-EGF脱落机制。在该系统中，表达α2AAR的“供体细胞”与缺乏α2AAR的“受体细胞”共培养。每个群体都表达一种独特的表位标记的ERK2蛋白，从而能够选择性地测量供体和受体细胞中的ERK激活。用α2AR选择性激动剂UK14304刺激可迅速增加供体和受体细胞中ERK2的磷酸化。受体细胞反应对EGFR和HB-EGF的抑制剂敏感，表明它是由表达α2AAR的供体细胞释放HB-EGF所致。用各种化学抑制剂和显性抑制突变体进行的实验表明，α2AAR刺激后ERK级联的EGFR依赖性激活在HB-EGF脱落和EGFR激活的上游需要Gβγ亚基，在下游需要发动蛋白依赖性内吞作用，而Src激酶活性对于HB-EGF的释放和HB-EGF介导的ERK2磷酸化都是必需的。
