Jiang Yong-Feng, Zhang Zaixiang, Kifor Olga, Lane Charles R, Quinn Stephen J, Bai Mei
Endocrine-Hypertension Division, Department of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2002 Dec 27;277(52):50543-9. doi: 10.1074/jbc.M205798200. Epub 2002 Oct 29.
The extracellular calcium (Ca(2+)(o))-sensing receptor (CaR) activates Ca(2+) influx independent of the release of intracellular Ca(2+) stores. The latter can be negatively regulated by protein kinase C (PKC) through phosphorylation of Thr-888 of the CaR. In this study, we substituted Thr-888 with various amino acid residues or a stop codon to understand how PKC phosphorylation of the CaR inhibits receptor-mediated release of intracellular Ca(2+) stores. Substitutions of Thr-888 with hydrophobic and hydrophilic amino acid residues had various effects on CaR-mediated release of intracellular Ca(2+) stores as well as activation of Ca(2+) influx. Several point mutations, such as T888D, had marked negative effects on CaR-mediated release of intracellular Ca(2+) stores but not on phorbol myristate acetate-insensitive activation of Ca(2+) influx. Presumably, the negatively charged aspartate mimics phospho-threonine. Interestingly, truncating the receptor at 888 had an even more pronounced negative effect on CaR-elicited release of intracellular Ca(2+) stores without significantly affecting CaR-mediated activation of Ca(2+) influx. Therefore, truncation at position 888 of the CaR affects the activity of the receptor in a manner that resembles PKC phosphorylation of the CaR. This in turn suggests that PKC phosphorylation of the CaR prevents G protein subtypes from interacting with the region of the receptor critical for releasing Ca(2+) stores, which is missing in the truncated receptor.
细胞外钙（Ca(2+)(o)）-传感受体（CaR）激活Ca(2+)内流，且不依赖于细胞内Ca(2+)储存库的释放。后者可通过蛋白激酶C（PKC）对CaR的苏氨酸-888进行磷酸化而受到负调控。在本研究中，我们将苏氨酸-888替换为各种氨基酸残基或终止密码子，以了解CaR的PKC磷酸化如何抑制受体介导的细胞内Ca(2+)储存库的释放。用疏水和亲水氨基酸残基替换苏氨酸-888对CaR介导的细胞内Ca(2+)储存库的释放以及Ca(2+)内流的激活有不同影响。几个点突变，如T888D，对CaR介导的细胞内Ca(2+)储存库的释放有显著负面影响，但对佛波酯肉豆蔻酸酯乙酸盐不敏感的Ca(2+)内流激活没有影响。据推测，带负电荷的天冬氨酸模拟了磷酸化的苏氨酸。有趣的是，在888位截断受体对CaR引发的细胞内Ca(2+)储存库的释放有更明显的负面影响，而对CaR介导的Ca(2+)内流激活没有显著影响。因此，在CaR的888位截断会以类似于CaR的PKC磷酸化的方式影响受体的活性。这反过来表明，CaR的PKC磷酸化阻止了G蛋白亚型与受体中对释放Ca(2+)储存库至关重要的区域相互作用，而该区域在截断的受体中缺失。
