Stitham Jeremiah, Gleim Scott R, Douville Karen, Arehart Eric, Hwa John
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
J Biol Chem. 2006 Dec 1;281(48):37227-36. doi: 10.1074/jbc.M604042200. Epub 2006 Oct 2.
Prostacyclin plays important roles in vascular homeostasis, promoting vasodilatation and inhibiting platelet thrombus formation. Previous studies have shown that three of six cytoplasmic cysteines, particularly those within the C-terminal tail, serve as important lipidation sites and are differentially conjugated to palmitoyl and isoprenyl groups (Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947-6958). Here we report distinctive roles for extracellular- and transmembrane-located cysteine residues in human prostacyclin receptor structure-function. Within the extracellular domain, all cysteines (4 of 4) appear to be involved in disulfide bonding interactions (i.e. a highly conserved Cys-92-Cys-170 bond and a putative non-conserved Cys-5-Cys-165 bond), and within the transmembrane (TM) region there are several cysteines (3 of 8) that maintain critical hydrogen bonding interactions (Cys-118 (TMIII), Cys-251 (TMVI), and Cys-202 (TMV)). This study highlights the necessity of sulfhydryl (SH) groups in maintaining the structural integrity of the human prostacyclin receptor, as 7 of 12 extracellular and transmembrane cysteines studied were found to be differentially indispensable for receptor binding, activation, and/or trafficking. Moreover, these results also demonstrate the versatility and reactivity of these cysteine residues within different receptor environments, that is, extracellular (disulfide bonds), transmembrane (H-bonds), and cytoplasmic (lipid conjugation).
前列环素在血管稳态中发挥着重要作用，可促进血管舒张并抑制血小板血栓形成。先前的研究表明，六个胞质半胱氨酸中的三个，尤其是C末端尾巴中的那些，作为重要的脂化位点，与棕榈酰基和异戊二烯基有不同的共轭作用（Miggin, S. M., Lawler, O. A., and Kinsella, B. T. (2003) J. Biol. Chem. 278, 6947 - 6958）。在此，我们报告了人前列环素受体结构功能中位于细胞外和跨膜的半胱氨酸残基的独特作用。在细胞外结构域内，所有半胱氨酸（4个中的4个）似乎都参与二硫键相互作用（即高度保守的Cys - 92 - Cys - 170键和一个假定的非保守Cys - 5 - Cys - 165键），并且在跨膜（TM）区域有几个半胱氨酸（8个中的3个）维持关键的氢键相互作用（Cys - 118（TMIII）、Cys - 251（TMVI）和Cys - 202（TMV））。这项研究强调了巯基（SH）基团在维持人前列环素受体结构完整性方面的必要性，因为在所研究的12个细胞外和跨膜半胱氨酸中，有7个被发现对于受体结合、激活和/或运输有不同程度的不可或缺性。此外，这些结果还证明了这些半胱氨酸残基在不同受体环境中的多功能性和反应性，即细胞外（二硫键）、跨膜（氢键）和胞质（脂质共轭）。
