G蛋白偶联受体激酶结合蛋白1的SHD结构域对成骨细胞内局部黏附激酶活性的影响

目的 探讨G蛋白偶联受体激酶结合蛋白1(GITl)的SHD结构域对成骨细胞内局部黏附激酶(FAK)活性的影响,并分析其机制.方法 取1～2dSD大鼠颅盖骨进行成骨细胞原代培养,传至第3代.体外构建野生型GIT1和删除了SHD结构域的GIT1的质粒并组成慢病毒.血小板衍生生长因子(PDGF)刺激成骨细胞后,采用免疫共沉淀法检测GIT1、删除了SHD结构域的GIT1与FAK的相互关系,采用免疫荧光法检测GIT1和FAK在成骨细胞内的位置、删除了SHD结构域的GIT1对成骨细胞内FAK位置和活性的影响.结果 PDGF刺激成骨细胞后,GIT1与FAK的相互结合明显增加,且随着时间的增加,结合量逐渐增加(F=293.105,P=0.000).免疫荧光染色结果表明:PDGF刺激后,成骨细胞局部黏附内活性形式的FAK(FAK酪氨酸397磷酸化)明显增加.PDGF刺激成骨细胞后,删除了SHD结构域的GIT1与FAK的结合明显被抑制,且随着时间的增加,二者的相互关系无明显变化(F=0.322,P=0.737);删除了SHD结构域的GIT1抑制成骨细胞局部黏附内FAK酪氨酸397的磷酸化,同时降低FAK在局部黏附内的表达.结论 删除了SHD结构域的GIT1通过降低与FAK的相互结合,从而抑制成骨细胞局部黏附内FAK酪氨酸397的磷酸化,影响成骨细胞的功能.

Function of Spa2 homplogy domian in G protein coupled receptor kinase interacting protein 1 on focal adhesion kinase activation

Objective To study function and mechanism of the Spa2 homplogy domian(SHD)of G protein coupled receptor kinase interacting protein 1(GIT1)on focal adhesion kinase(FAK)activation in rat osteoblasts.Methods Osteoblasts were prepared from the calvaria of SD rats 1 to 2 days old.The third generation of osteoblasts was treated with platelet-derived growth factor(PDGF)to detect the relationship between GIT1 and FAK.Localizations of GIT1 and FAK were determined by immunofluorescent staining with or without PDGF stimulation.We made GIT1 and GIT1(del SHD)lentiviruses to detect the relationship between GIT1(del SHD)and FAK by imnunoprecipitation.The FAK activation was checked in osteoblastic cells infected with GIT1 and GIT1(del SHD)lentiviruses by immunofluorescent staining.Results The association between GIT1 and FAK was increased after PDGF stimulation.GIT1 and FAK were localized in focal adhesion in osteoblastic cells after PDGF stimulation.The interaction between GIT1(del SHD)and FAK was not changed after PDGF stimulation(F =0.322,P =0.737).The GIT1(del SHD)inhibited the FAK activation in focal adhesion in osteoblastic cells.Conclusion GIT1(del SHD)can inhibit the association with FAK and decrease the FAK activation in focal adhesion in osteoblast cells after PDGF stimulation,affecting the capacity of osteoblasts.