Shimizu Naoto, Dean Thomas, Tsang Janet C, Khatri Ashok, Potts John T, Gardella Thomas J
Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, USA.
J Biol Chem. 2005 Jan 21;280(3):1797-807. doi: 10.1074/jbc.M408270200. Epub 2004 Nov 17.
Current antagonists for the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (PTHR) are N-terminally truncated or N-terminally modified analogs of PTH(1-34) or PTHrP(1-34) and are thought to bind predominantly to the N-terminal extracellular (N) domain of the receptor. We hypothesized that ligands that bind only to PTHR region comprised of the extracellular loops and seven transmembrane helices (the juxtamembrane or J domain) could also antagonize the PTHR. To test this, we started with the J domain-selective agonists [Gln(10),Ala(12),Har(11),Trp(14),Arg(19) (M)]PTH(1-21), [M]PTH(1-15), and [M]PTH(1-14), and introduced substitutions at positions 1-3 that were predicted to dissociate PTHR binding and cAMP signaling activities. Strong dissociation was observed with the tri-residue sequence diethylglycine (Deg)(1)-para-benzoyl-l-phenylalanine (Bpa)(2)-Deg(3). In HKRK-B7 cells, which express the cloned human PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21), [Deg(1,3),Bpa(2),M]PTH(1-15), and [Deg(1,3),Bpa(2),M]PTH(1-14) fully inhibited (IC(50)s = 100-700 nm) the binding of (125)I-[alpha-aminoisobutyric acid(1,3),M]PTH(1-15) and were severely defective for stimulating cAMP accumulation. In ROS 17/2.8 cells, which express the native rat PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) antagonized the cAMP-agonist action of PTH(1-34), as did PTHrP(5-36) (IC(50)s = 0.7 microm, 2.6 microm, and 36 nm, respectively). In COS-7 cells expressing PTHR-delNt, which lacks the N domain of the receptor, [Deg(1,3),Bpa(2), M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) inhibited the agonist actions of [alpha-aminoisobutyric acid(1,3)]PTH(1-34) and [M]PTH(1-14) (IC(50)s approximately 1 microm), whereas PTHrP(5-36) failed to inhibit. [Deg(1,3),Bpa(2),M]PTH(1-14) inhibited the constitutive cAMP-signaling activity of PTHR-tether-PTH(1-9), in which the PTH(1-9) sequence is covalently linked to the PTHR J domain, as well as that of PTHR(cam)H223R. Thus, the J-domain-selective N-terminal PTH fragment analogs can function as antagonists as well as inverse agonists for the PTHR. The new ligands described should be useful for further studies of the ligand binding and activation mechanisms that operate in the critical PTHR J domain.
目前，甲状旁腺激素（PTH）/PTH相关蛋白（PTHrP）受体（PTHR）的拮抗剂是PTH(1 - 34)或PTHrP(1 - 34)的N端截短或N端修饰类似物，据认为它们主要与受体的N端细胞外（N）结构域结合。我们推测，仅与由细胞外环和七个跨膜螺旋组成的PTHR区域（近膜或J结构域）结合的配体也可能拮抗PTHR。为了验证这一点，我们从J结构域选择性激动剂[Gln(10),Ala(12),Har(11),Trp(14),Arg(19) (M)]PTH(1 - 21)、[M]PTH(1 - 15)和[M]PTH(1 - 14)入手，并在预测会使PTHR结合和cAMP信号传导活性解离的1 - 3位引入取代基。观察到三残基序列二乙基甘氨酸（Deg）(1)-对苯甲酰基-L-苯丙氨酸（Bpa）(2)-Deg(3)有强烈的解离作用。在表达克隆的人PTHR的HKRK - B7细胞中，[Deg(1,3),Bpa(2),M]PTH(1 - 21)、[Deg(1,3),Bpa(2),M]PTH(1 - 15)和[Deg(1,3),Bpa(2),M]PTH(1 - 14)完全抑制（IC50 = 100 - 700 nM）(125)I-[α-氨基异丁酸(1,3),M]PTH(1 - 15)的结合，并且在刺激cAMP积累方面存在严重缺陷。在表达天然大鼠PTHR的ROS 17/2.8细胞中，[Deg(1,3),Bpa(2),M]PTH(1 - 21)和[Deg(1,3),Bpa(2),M]PTH(1 - 15)拮抗PTH(1 - 34)的cAMP激动剂作用，PTHrP(5 - 36)也是如此（IC50分别为0.7 μM、2.6 μM和36 nM）。在表达缺乏受体N结构域的PTHR - delNt的COS - 7细胞中，[Deg(1,3),Bpa(2), M]PTH(1 - 21)和[Deg(1,3),Bpa(2),M]PTH(1 - 15)抑制[α-氨基异丁酸(1,3)]PTH(1 - 34)和[M]PTH(1 - 14)的激动剂作用（IC50约为1 μM），而PTHrP(5 - 36)未能抑制。[Deg(1,3),Bpa(2),M]PTH(1 - 14)抑制PTHR - tether - PTH(1 - 9)的组成性cAMP信号传导活性，其中PTH(1 - 9)序列与PTHR J结构域共价连接，以及PTHR(cam)H223R的活性。因此，J结构域选择性N端PTH片段类似物可作为PTHR的拮抗剂以及反向激动剂发挥作用。所描述的新配体应有助于进一步研究在关键的PTHR J结构域中起作用的配体结合和激活机制。
