Selective phosphorylation of threonine residues defines GPR84-arrestin interactions of biased ligands.
Selective phosphorylation of threonine residues defines GPR84-arrestin interactions of biased ligands.
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DOI：
10.1016/j.jbc.2022.101932
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发表时间：
2022-05
影响因子：
4.8
通讯作者：
Milligan, Graeme
中科院分区：
文献类型：
--
作者：
Marsango, Sara;Ward, Richard J.;Jenkins, Laura;Butcher, Adrian J.;Al Mahmud, Zobaer;Dwomoh, Louis;Nagel, Falko;Schulz, Stefan;Tikhonova, Irina G.;Tobin, Andrew B.;Milligan, Graeme
GPR 84是一种免疫细胞表达的促炎受体，目前被评估为包括纤维化和炎症性肠病在内的疾病的治疗靶点。尽管先前显示正构GPR 84激活剂2-HTP和6-OAU促进其与arrestin-3的相互作用，但G蛋白偏向性激动剂DL-175没有。在这里，我们表明，替换GPR 84的i环3内的所有21个丝氨酸和苏氨酸残基，而不是C-末端尾部的两个丝氨酸，消除了[32 P]的掺入，并大大降低了由2-HTP促进的受体-arrestin-3相互作用。GPR 84在残基Ser 221和Ser 224上组成性磷酸化，而各种其它氨基酸响应于2-HTP而磷酸化。与此一致，能够识别pSer 221/pSer 224的抗血清识别来自用和不用活化剂处理的细胞的GPR 84，而能够识别pThr 263/pThr 264的抗血清在暴露于2-HTP而不是DL-175后仅识别GPR 84。两种不同的GPR 84拮抗剂以及G蛋白偶联受体激酶2/3的抑制阻止了pThr 263/pThr 264的磷酸化，但这两种策略都不影响Ser 221/Ser 224的组成性磷酸化。此外，将残基Thr 263和Thr 264突变为丙氨酸产生的GPR 84变体也限制了2-HT诱导的与抑制蛋白-2和-3的相互作用。相比之下，该突变体在降低cAMP水平的能力方面不受影响。综上所述，这些结果定义了一个关键的苏氨酸残基对，仅受GPR 84小分子激活剂亚组和GRK 2/3的调节，其定义了与抑制蛋白的有效相互作用，并提供了监测GPR 84磷酸化和功能状态的新工具。
GPR84 is an immune cell–expressed, proinflammatory receptor currently being assessed as a therapeutic target in conditions including fibrosis and inflammatory bowel disease. Although it was previously shown that the orthosteric GPR84 activators 2-HTP and 6-OAU promoted its interactions with arrestin-3, a G protein–biased agonist DL-175 did not. Here, we show that replacement of all 21 serine and threonine residues within i-loop 3 of GPR84, but not the two serines in the C-terminal tail, eliminated the incorporation of [32P] and greatly reduced receptor–arrestin-3 interactions promoted by 2-HTP. GPR84 was phosphorylated constitutively on residues Ser221 and Ser224, while various other amino acids are phosphorylated in response to 2-HTP. Consistent with this, an antiserum able to identify pSer221/pSer224 recognized GPR84 from cells treated with and without activators, whereas an antiserum able to identify pThr263/pThr264 only recognized GPR84 after exposure to 2-HTP and not DL-175. Two distinct GPR84 antagonists as well as inhibition of G protein–coupled receptor kinase 2/3 prevented phosphorylation of pThr263/pThr264, but neither strategy affected constitutive phosphorylation of Ser221/Ser224. Furthermore, mutation of residues Thr263 and Thr264 to alanine generated a variant of GPR84 also limited in 2-HTP–induced interactions with arrestin-2 and -3. By contrast, this mutant was unaffected in its capacity to reduce cAMP levels. Taken together, these results define a key pair of threonine residues, regulated only by subsets of GPR84 small molecule activators and by GRK2/3 that define effective interactions with arrestins and provide novel tools to monitor the phosphorylation and functional status of GPR84.
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DOI：
10.1016/j.jbc.2022.101655
发表时间：
2022-03
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Divorty N;Jenkins L;Ganguly A;Butcher AJ;Hudson BD;Schulz S;Tobin AB;Nicklin SA;Milligan G
通讯作者：
Milligan G
影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
7.3
作者：
通讯作者：
--
作为有效 GPR84 激动剂的 2-烷基嘧啶-4,6-二醇和 6-烷基吡啶-2,4-二醇的设计和合成。
影响因子：
4.2
作者：
Liu, Yang;Zhang, Qing;Nan, Fa-Jun
通讯作者：
Nan, Fa-Jun
影响因子：
4
作者：
Lucy, Daniel;Purvis, Gareth S. D.;Russell, Angela J.
通讯作者：
Russell, Angela J.