Keating M T, Escobedo J A, Williams L T
University of California, Department of Medicine, San Francisco.
J Biol Chem. 1988 Sep 15;263(26):12805-8.
The effect of ligand binding on platelet-derived growth factor (PDGF) receptor conformation was examined using peptide antibodies directed against specific receptor domains. Antiserum 83, which was directed to the receptor's carboxyl terminus (residues 934-951), preferentially immunoprecipitated the ligand-activated form of the PDGF receptor from 35S-labeled BALB/c 3T3 cells. By contrast, two antisera directed against other receptor sequences precipitated unactivated and activated receptors equally well. Denatured receptors were recognized equally by all antisera, even 83. Thus, ligand activation caused a change in PDGF receptor conformation that enhanced accessibility of the antibody to the carboxyl terminus. The activated receptor conformation was induced by three different forms of PDGF (AA and BB homodimers and AB heterodimers) and was reversed by suramin, a polyanionic compound that dissociates PDGF from the receptor. The inhibitory effect of suramin on receptor conformation was abolished by the phosphatase inhibitor, sodium orthovanadate, suggesting that receptor phosphorylation mediated the conformational change. In a cell-free assay, the change in receptor conformation was induced by PDGF only in the presence of ATP and was inhibited by adenyl-5'-yl imidodiphosphate, a nonhydrolyzable analog of ATP. The functional significance of receptor conformation was examined in Chinese hamster ovary fibroblasts transfected with wild-type or mutated forms of the PDGF receptor. When receptor tyrosine kinase activity was abolished by a mutation of the ATP binding site the receptor no longer underwent PDGF-induced conformational change and did not mediate PDGF-induced mitogenesis even though 125I-PDGF binding was normal. These findings show that ligand binding elicits a phosphorylation-dependent change in PDGF receptor conformation that may be important for receptor function.
使用针对特定受体结构域的肽抗体，研究了配体结合对血小板衍生生长因子（PDGF）受体构象的影响。抗血清83针对受体的羧基末端（第934 - 951位氨基酸残基），它优先从35S标记的BALB/c 3T3细胞中免疫沉淀配体激活形式的PDGF受体。相比之下，另外两种针对受体其他序列的抗血清对未激活和激活的受体沉淀效果相同。所有抗血清对变性受体的识别效果相同，包括抗血清83。因此，配体激活导致PDGF受体构象发生变化，增强了抗体对羧基末端的可及性。三种不同形式的PDGF（AA和BB同二聚体以及AB异二聚体）均可诱导激活的受体构象，而多阴离子化合物苏拉明可使PDGF与受体解离，从而逆转这种构象。磷酸酶抑制剂原钒酸钠可消除苏拉明对受体构象的抑制作用，这表明受体磷酸化介导了构象变化。在无细胞测定中，仅在ATP存在的情况下，PDGF才能诱导受体构象变化，而ATP的非水解类似物腺苷-5'-亚氨基二磷酸可抑制这种变化。在中国仓鼠卵巢成纤维细胞中，转染野生型或突变型PDGF受体后，研究了受体构象的功能意义。当ATP结合位点发生突变使受体酪氨酸激酶活性丧失时，受体不再发生PDGF诱导的构象变化，也不介导PDGF诱导的有丝分裂，尽管125I - PDGF结合正常。这些发现表明，配体结合引发了PDGF受体构象的磷酸化依赖性变化，这可能对受体功能很重要。
