Angelova Krassimira, Puett David
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
Endocrine. 2002 Nov;19(2):147-54. doi: 10.1385/ENDO:19:2:147.
The glycoprotein hormone receptors-luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), and thyroid-stimulating hormone receptor (TSHR)--are G-protein-coupled receptors with an invariant 10-amino acid residue sequence in the ectodomain proximal to transmembrane helix 1. A Glu-Asp, located at the midpoint of this conserved sequence, has been suggested to be important in ligand-mediated signaling of LHR and/or receptor expression or stability, but not binding. One goal of this study was to expand the studies on LHR and determine whether the invariant Glu and Asp residues were functional in FSHR and TSHR as well. Another goal was to investigate systematically the role of ionic strength, particularly Na+, which appears to have enigmatic functions in the three receptors regarding ligand binding and receptor activation, and to ascertain whether any of the purported effects of Na+ could involve the conserved pair of acidic side chains in the ectodomain. COS-7 cells were transiently transfected with cDNAs to the wild-type (WT) receptor (rat) and identical single and double mutants of each (Glu --> Ala, Asp; Asp --> Ala, Glu; and Glu-Asp--> Asp-Glu), followed by characterization of cognate ligand binding and signaling (basal and hormone mediated) in two commonly used buffer systems: Waymouth's medium, containing a near-physiologic concentration of Na+ (132 mM); a low ionic strength buffer with a 1 mM concentration of Na+. The three receptors exhibited differential responses to mutagenesis and the two buffers. Notably, a comparison of basal cyclic adenosine monophosphate (cAMP) production showed that the buffer of lower ionic strength resulted in increased basal cAMP production in WT TSHR but not LHR and FSHR; that the maximal ligand-mediated cAMP production was greatest in the buffer of higher ionic strength for the three WT receptors; that functionality of the conserved Glu and Asp residues in ligand-mediated signaling was buffer dependent in LHR, whereas it did not appear to be particularly important in FSHR and TSHR signaling; and that apparent ligand binding in WT and mutant TSHRs seemed to be particularly diminished in the buffer of higher ionic strength. These results demonstrate that identical amino acid residues in homologous receptors can exhibit distinct functions; moreover, the role of ionic strength (Na+) on signaling differs in the three receptors.
糖蛋白激素受体——促黄体生成素受体（LHR）、促卵泡激素受体（FSHR）和促甲状腺激素受体（TSHR）——是G蛋白偶联受体，在靠近跨膜螺旋1的胞外域有一个不变的10个氨基酸残基序列。位于这个保守序列中点的一个谷氨酸-天冬氨酸（Glu-Asp），被认为在LHR的配体介导信号传导和/或受体表达或稳定性方面很重要，但在结合方面不重要。本研究的一个目标是扩展对LHR的研究，并确定不变的Glu和Asp残基在FSHR和TSHR中是否也有功能。另一个目标是系统地研究离子强度，特别是Na+的作用，Na+在这三种受体的配体结合和受体激活方面似乎具有神秘的功能，并确定Na+的任何所谓作用是否可能涉及胞外域中保守的酸性侧链对。用野生型（WT）受体（大鼠）的cDNA以及每种受体相同的单突变体和双突变体（Glu→Ala、Asp；Asp→Ala、Glu；以及Glu-Asp→Asp-Glu）瞬时转染COS-7细胞，然后在两种常用缓冲系统中表征同源配体结合和信号传导（基础和激素介导）：含有接近生理浓度Na+（132 mM）的Waymouth培养基；Na+浓度为1 mM的低离子强度缓冲液。这三种受体对诱变和两种缓冲液表现出不同的反应。值得注意的是，基础环磷酸腺苷（cAMP）产生的比较表明，较低离子强度的缓冲液导致WT TSHR中基础cAMP产生增加，但LHR和FSHR中没有；对于三种WT受体，最大配体介导的cAMP产生在较高离子强度的缓冲液中最大；在LHR中，保守的Glu和Asp残基在配体介导信号传导中的功能依赖于缓冲液，而在FSHR和TSHR信号传导中似乎不是特别重要；并且WT和突变型TSHR中的表观配体结合在较高离子强度的缓冲液中似乎特别减少。这些结果表明，同源受体中相同的氨基酸残基可以表现出不同的功能；此外，离子强度（Na+）对信号传导的作用在这三种受体中有所不同。
