Hauache O M, Hu J, Ray K, Xie R, Jacobson K A, Spiegel A M
Metabolic Diseases Branch NIDDK, NIH, Bethesda, Maryland 20892, USA.
Endocrinology. 2000 Nov;141(11):4156-63. doi: 10.1210/endo.141.11.7753.
Naturally occurring mutations identified in subjects with autosomal dominant hypocalcemia (ADH) and the calcimimetic compound, R-568, have both been reported to increase Ca2+ sensitivity of the Ca2+ receptor (CaR). To gain insight into their mechanism of action, we studied interactions between four different ADH mutations located in the amino-terminal extracellular domain (ECD) and R-568. We found that R-568 increased the sensitivity of three of the ADH mutant receptors, but the Leu125Pro mutant appeared to be maximally left-shifted in that neither R-568 addition nor combining other ADH mutations with Leu125Pro gave increases in sensitivity comparable to those seen with the three other ADH mutations studied. We also made use of truncation and deletion mutants of the CaR and CaR/metabotropic glutamate receptor type 1 (mGluR1) chimeras to study both the site of action of R-568 and the effect of the Leu125Pro activating mutation. R-568 was effective in receptor constructs containing the seven transmembrane domain (7TM) of the CaR, but not in those containing the mGluR1 7TM. R-568, moreover, imparted Ca2+ responsiveness to CaR constructs lacking all or part of the CaR ECD. The Leu125Pro mutation in contrast conferred no or minimal increase in Ca2+ responsiveness to CaR constructs lacking part of the CaR ECD but showed a striking increase in basal activity in the context of chimeras containing an mGluR1 7TM. Our results localize the site of action of NPS-568 specifically to the CaR 7TM. Our results with the Leu125Pro mutant, furthermore, suggest that the mGluR1 7TM domain may be more permissive for activation than the 7TM domain of the CaR.
据报道，在患有常染色体显性低钙血症（ADH）的受试者中发现的自然发生的突变以及拟钙剂化合物R-568，均可增加钙离子受体（CaR）对Ca2+的敏感性。为深入了解它们的作用机制，我们研究了位于氨基末端细胞外结构域（ECD）的四种不同ADH突变与R-568之间的相互作用。我们发现，R-568增加了三种ADH突变型受体的敏感性，但Leu125Pro突变体似乎向左最大程度地偏移，因为添加R-568或使其他ADH突变与Leu125Pro组合均未使敏感性增加到与所研究的其他三种ADH突变相当的程度。我们还利用CaR的截短和缺失突变体以及CaR/代谢型谷氨酸受体1（mGluR1）嵌合体来研究R-568的作用位点以及Leu125Pro激活突变的影响。R-568在含有CaR七次跨膜结构域（7TM）的受体构建体中有效，但在含有mGluR1 7TM的构建体中无效。此外，R-568赋予缺乏全部或部分CaR ECD的CaR构建体Ca2+反应性。相比之下，Leu125Pro突变对缺乏部分CaR ECD的CaR构建体的Ca2+反应性没有增加或增加极少，但在含有mGluR1 7TM的嵌合体中，其基础活性显著增加。我们的结果将NPS-568的作用位点特异性定位于CaR 7TM。此外，我们对Leu125Pro突变体的研究结果表明，mGluR1 7TM结构域可能比CaR的7TM结构域对激活更具耐受性。
