Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor.
Agonist-induced phosphorylation of orthologues of the orphan receptor GPR35 functions as an activation sensor.
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DOI：
10.1016/j.jbc.2022.101655
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发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Milligan G
中科院分区：
文献类型：
--
作者：
Divorty N;Jenkins L;Ganguly A;Butcher AJ;Hudson BD;Schulz S;Tobin AB;Nicklin SA;Milligan G
G蛋白偶联受体35（GPR 35）的特征很差，但已被揭示在包括下肠道炎症和疼痛在内的领域中具有多种作用。新试剂和工具的开发将大大增强对健康和疾病中GPR 35功能的分析。在这里，我们使用质谱法，诱变，和[32 P]正磷酸标记，以确定所有五个羟基氨基酸的C-末端尾的人GPR 35 a成为磷酸化的受体的激动剂占用的反应，除了Ser 294，这些都有助于与rectin-3，抑制进一步的G蛋白偶联受体信号的相互作用。我们发现Ser 303是这种相互作用的关键;对应于人GPR 35 a残基303的丝氨酸也在小鼠和大鼠GPR 35的arrestin-3相互作用中起主导作用。我们还证明了人GPR 35 a和小鼠GPR 35的完全磷酸化位点缺陷突变体未能有效地与抑制蛋白-3相互作用，并且人磷酸化缺陷变体在响应激动剂处理时不会从细胞表面内化。即使在稳定表达GPR 35的物种直向同源物的细胞中，也确定大部分表达的蛋白质是不成熟的。最后，磷酸化位点特异性抗血清靶向的区域涵盖Ser 303在人类（Ser 301在小鼠）GPR 35 a确定只有成熟形式的GPR 35，并提供了有效的传感器的激活状态的受体在免疫印迹和免疫细胞化学研究。这种抗血清可能是有用的工具，以评估药物发现和目标验证程序中的目标接合。
G protein-coupled receptor 35 (GPR35) is poorly characterized but nevertheless has been revealed to have diverse roles in areas including lower gut inflammation and pain. The development of novel reagents and tools will greatly enhance analysis of GPR35 functions in health and disease. Here, we used mass spectrometry, mutagenesis, and [32P] orthophosphate labeling to identify that all five hydroxy-amino acids in the C-terminal tail of human GPR35a became phosphorylated in response to agonist occupancy of the receptor and that, apart from Ser294, each of these contributed to interactions with arretin-3, which inhibits further G protein-coupled receptor signaling. We found that Ser303 was key to such interactions; the serine corresponding to human GPR35a residue 303 also played a dominant role in arrestin-3 interactions for both mouse and rat GPR35. We also demonstrated that fully phospho-site–deficient mutants of human GPR35a and mouse GPR35 failed to interact effectively with arrestin-3, and the human phospho-deficient variant was not internalized from the surface of cells in response to agonist treatment. Even in cells stably expressing species orthologues of GPR35, a substantial proportion of the expressed protein(s) was determined to be immature. Finally, phospho-site–specific antisera targeting the region encompassing Ser303 in human (Ser301 in mouse) GPR35a identified only the mature forms of GPR35 and provided effective sensors of the activation status of the receptors both in immunoblotting and immunocytochemical studies. Such antisera may be useful tools to evaluate target engagement in drug discovery and target validation programs.
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DOI：
10.1074/jbc.m116.754887
发表时间：
2016-12-30
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Alvarez-Curto E;Inoue A;Jenkins L;Raihan SZ;Prihandoko R;Tobin AB;Milligan G
通讯作者：
Milligan G
影响因子：
64.5
作者：
Latorraca NR;Masureel M;Hollingsworth SA;Heydenreich FM;Suomivuori CM;Brinton C;Townshend RJL;Bouvier M;Kobilka BK;Dror RO
通讯作者：
Dror RO
影响因子：
64.8
作者：
Latorraca NR;Wang JK;Bauer B;Townshend RJL;Hollingsworth SA;Olivieri JE;Xu HE;Sommer ME;Dror RO
通讯作者：
Dror RO
影响因子：
4.1
作者：
Jenkins, Laura;Brea, Jose;Milligan, Graeme
通讯作者：
Milligan, Graeme
DOI：
10.1007/164_2015_42
发表时间：
2017-01-01
期刊：
NON-CANONICAL CYCLIC NUCLEOTIDES
影响因子：
--
作者：
Grundmann, Manuel;Kostenis, Evi
通讯作者：
Kostenis, Evi