Regions in the G protein γ subunit important for interaction with receptors and effectors
Regions in the G protein γ subunit important for interaction with receptors and effectors
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DOI：
10.1124/mol.105.018994
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发表时间：
2006-03-01
影响因子：
3.6
通讯作者：
Garrison, JC
中科院分区：
文献类型：
--
作者：
Myung, CS;Lim, WK;Garrison, JC
与含有γ（2）亚基的二聚体相比，含有γ（11）或γ（1）亚基的G β γ二聚体在调节效应物的能力方面通常较不强效和有效。为了探索影响β-γ二聚体活性的γ亚基区域，我们从γ（1）和γ（2）亚基构建了8个嵌合γ亚基。制备了两个嵌合体，其中γ（1）和γ（2）的N-末端区域被交换，并且其中两个C-末端区域被转座。制备另一组嵌合体，其中嵌合体的CAAX基序被改变为用不同的异戊烯基直接修饰。所有8种γ嵌合体均在Sf 9细胞中表达β（1）亚基，纯化G β γ二聚体，然后在体外测定它们与G α（i1）亚基结合、将G α（i1）偶联至A1腺苷受体、刺激磷脂酶C-β和调节I型或II型腺苷酸环化酶的能力。含有用香叶基香叶基脂质修饰的γ（2）亚基的C末端序列的二聚体对G（i1）α具有最高的亲和力（范围，0.5-1.2 nM），并且在将G（i1）α亚基与受体偶联时最有效。这些二聚体在刺激磷脂酰肌醇特异性磷脂酶C-β亚型和抑制I型腺苷酸环化酶方面最有效。相比之下，含有γ（2）亚基的N-末端序列和香叶基香叶基的β-γ二聚体在激活II型腺苷酸环化酶方面最有效。结果表明，γ亚基的N-和C-末端区域赋予受体和效应物相互作用的特异性。
G beta gamma dimers containing the gamma(11) or gamma(1) subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the gamma(2) subunit. To explore the regions of the gamma subunit that affect the activity of the beta gamma dimer, we constructed eight chimeric gamma subunits from the gamma(1) and gamma(2) subunits. Two chimeras were made in which the N-terminal regions of gamma(1) and gamma(2) were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight gamma chimeras were expressed in Sf9 cells with the beta(1) subunit, G beta gamma dimers were purified, and then they were assayed in vitro for their ability to bind to the G alpha(i1) subunit, to couple G alpha(i1) to the A1 adenosine receptor, to stimulate phospholipase C-beta, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the gamma(2) subunit modified with the geranylgeranyl lipid had the highest affinity for G(i1)alpha (range, 0.5-1.2 nM) and were most effective at coupling the G(i1)alpha subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C-beta isoform and inhibiting type I adenyl cyclase. In contrast, beta gamma dimers containing the N-terminal sequence of the gamma(2) subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N- and C-terminal regions of the gamma subunit impart specificity to receptor and effector interactions.