Mechanisms of ligand binding to the parathyroid hormone (PTH)/PTH-related protein receptor:: Selectivity of a modified PTH(1-15) Radioligand for GαS-coupled receptor conformations
Mechanisms of ligand binding to the parathyroid hormone (PTH)/PTH-related protein receptor:: Selectivity of a modified PTH(1-15) Radioligand for GαS-coupled receptor conformations
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DOI：
10.1210/me.2005-0349
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发表时间：
2006-04-01
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通讯作者：
Gardella, TJ
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文献类型：
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作者：
Dean, T;Linglart, A;Gardella, TJ
以甲状旁腺素片段类似物为放射性配体，探讨了配体与甲状旁腺素/甲状旁腺素受体(PTHR)结合的机制。特别是，修饰的氨基端片段类似物I-125-[AIB(1，3)，NLE(8)，Gln(10)，HomoArg(11)，Ala(12)，Trp(14)，Tyr(15)]rPTH(1-15)NH2，I-125-[AIB(1，3)，M]PTH(1-15)被用作放射性配体，我们推测它只与PTHR的膜旁部分结合，含有胞外环和跨膜螺旋。我们还使用了I-125-PTH(1-34)作为与PTHR的氨基末端胞外(N)和J结构域结合的放射性配体。从表达野生型或突变型PTHR的细胞中获得的细胞膜中检测到结合。我们发现，I-125[AIB(1，3)，M]PTH(1-15)与野生型PTHR的结合可被鸟苷5‘-O-(3-硫代)三磷酸(GTP-γS)强烈抑制(类似于90%)，而I-125-PTH(1-34)的结合仅被GTP-Gamma S轻度抑制(类似于25%)。而I-125-[AIB(1，3)，M]PTH(1-15)与其固有活性受体PTHR-H_223R的结合仅被GTP-γS轻度抑制(类似于20%)，与I-125-PTH(1-34)的结合一样。在缺乏Gα的细胞(S)通过GNAS基因敲除突变制备的膜中，未观察到I-125-[AIB(1，3)，M]PTH(1-15)的结合，但通过病毒转导异源表达Gα(S)，I-125-[AIB(1，3)，M]PTH(1-15)的结合恢复。I-125-PTH(1-34)与膜结合或不与Gα结合(S)。这些结果证实了I-125-[AIB(1，3)，M]PTH(1-15)只与PTHR的J结构域结合的假设。他们进一步表明，这种结合强烈依赖于受体与含有Gα(S)的异三聚体G蛋白的偶联，而I-125-PTH(1-34)的结合可以在没有这种偶联的情况下发生。因此，I-125-[AIB(1，3)，M]PTH(1-15)似乎是Gα(S)偶联的活化态PTHR构象的选择性探针。
Mechanisms of ligand binding to the PTH/PTHrP receptor (PTHR) were explored using PTH fragment analogs as radioligands in binding assays. In particular, the modified amino-terminal fragment analog, I-125-[Aib(1,3), Nle(8), Gln(10), homoarginine(11), Ala(12), Trp(14), Tyr(15)] rPTH(1 - 15) NH2, I-125-[Aib(1,3), M] PTH(1 - 15), was used as a radioligand that we hypothesized to bind solely to the juxtamembrane (J) portion of the PTHR containing the extracellular loops and transmembrane helices. We also employed I-125- PTH(1-34) as a radioligand that binds to both the amino-terminal extracellular (N) and J domains of the PTHR. Binding was examined in membranes derived from cells expressing either wild-type or mutant PTHRs. We found that the binding of I-125[ Aib(1,3), M] PTH(1 - 15) to the wild-type PTHR was strongly (similar to 90%) inhibited by guanosine 5'-O-(3-thio) triphosphate (GTP gamma S), whereas the binding of I-125- PTH( 1 - 34) was only mildly ( similar to 25%) inhibited by GTP gamma S. Of these two radioligands, only I-125[ Aib(1,3), M] PTH( 1 - 15) bound to PTHR-delNt, which lacks most of the receptor's N domain, and again this binding was strongly inhibited by GTP gamma S. Binding of I-125-[Aib(1,3), M] PTH(1 - 15) to the constitutively active receptor, PTHR-H223R, was only mildly ( similar to 20%) inhibited by GTP gamma S, as was the binding of I-125- PTH(1 - 34). In membranes prepared from cells lacking G alpha(s) via knockout mutation of Gnas, no binding of I-125-[Aib(1,3), M] PTH(1 - 15) was observed, but binding of I-125-[Aib(1,3), M] PTH( 1 - 15) was recovered by virally transducing the cells to heterologously express G alpha(s). I-125-PTH( 1 - 34) bound to the membranes with or without G alpha(s). The overall findings confirm the hypothesis that I-125-[ Aib(1,3), M] PTH( 1 - 15) binds solely to the J domain of the PTHR. They further show that this binding is strongly dependent on coupling of the receptor to G alpha(s)-containing heterotrimeric G proteins, whereas the binding of I-125- PTH( 1 - 34) can occur in the absence of such coupling. Thus, I-125-[ Aib(1,3), M] PTH( 1 - 15) appears to function as a selective probe of G alpha(s)-coupled, active-state PTHR conformations.