Hinson T K, Damodaran T V, Chen J, Zhang X, Qumsiyeh M B, Seldin M F, Quarles L D
Department of Medicine and Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Genomics. 1997 Oct 15;45(2):279-89. doi: 10.1006/geno.1997.4943.
The sensing of extracellular calcium is a general paradigm for regulating diverse cellular functions in many tissues. A calcium-sensing receptor (Casr) belonging to the metabotropic glutamate family of G-protein-coupled receptors (GPCR) that transduces the effects of extracellular calcium in the parathyroid gland as well as other tissues has been identified. The diversity of GPCR families and the recent finding of calcium sensing in cells lacking the known Casr suggest the existence of additional receptors related to Casr. By polymerase chain reaction (PCR) amplification and screening of genomic libraries, we have identified multiple Casr-related sequences (Casr-rs) in the mouse. Using primers designed to regions of the first and third intracellular loops of Casr, we initially PCR amplified a 497-bp Casr-related sequence (Casr-rs1) with high homology to Casr. The deduced protein sequence of Casr-rs1 is 63% similar and 40% identical to Casr over the available transmembrane region. We screened a mouse genomic library with a Casr-rs1 probe and identified two additional Casr-related sequences (Casr-rs2 and Casr-rs3). In the predicted transmembrane domain, Casr-rs2 and Casr-rs3 are 95% identical to Casr-rs1. We mapped Casr-rs1 to mouse Chromosome (Chr) 7 by interspecific backcross analysis, whereas the known Casr localizes to mouse Chr 16. By fluorescence in situ hybridization, Casr-rs2 also localized to mouse Chr 7 and Casr-rs3 mapped to mouse Chr 4. We were able to distinquish Casr-rs1 from Casr-rs2 by PCR using specific primers, suggesting that they are distinct genes clustered on Chr 7. By RT-PCR, we identified additional Casr-rs transcripts in mouse kidney, brain, testis, embryo, and MC3T3-E1 osteoblasts, but not in lung or liver. The homologous sequence in mouse kidney, embryo, and MC3T3-E1 osteoblasts, designated Casr-rs4, has a deduced amino acid sequence that is 100% similar and 97% identical to that of Casr-rs1. The sequence amplified from mouse brain, Casr-rs5, has a deduced protein sequence that is 96% similar and 92% identical to that of Casr-rs1. Our findings establish the existence of a novel multimembered family of Casr-related sequences in the mouse which may encode receptors that transduce responses to diverse extracellular cations.
细胞外钙的感知是调节许多组织中多种细胞功能的一般模式。已经鉴定出一种属于G蛋白偶联受体（GPCR）代谢型谷氨酸家族的钙敏感受体（Casr），它可转导甲状旁腺以及其他组织中细胞外钙的作用。GPCR家族的多样性以及最近在缺乏已知Casr的细胞中发现钙感知现象，表明存在与Casr相关的其他受体。通过聚合酶链反应（PCR）扩增和基因组文库筛选，我们在小鼠中鉴定出多个与Casr相关的序列（Casr-rs）。使用针对Casr第一和第三细胞内环区域设计的引物，我们最初通过PCR扩增出一个与Casr具有高度同源性的497 bp Casr相关序列（Casr-rs1）。在可用的跨膜区域，Casr-rs1的推导蛋白序列与Casr的相似度为63%，一致性为40%。我们用Casr-rs1探针筛选了小鼠基因组文库，并鉴定出另外两个与Casr相关的序列（Casr-rs2和Casr-rs3）。在预测的跨膜结构域中，Casr-rs2和Casr-rs3与Casr-rs1的一致性为95%。通过种间回交分析，我们将Casr-rs1定位到小鼠染色体（Chr）7上，而已知的Casr定位于小鼠Chr 16。通过荧光原位杂交，Casr-rs2也定位于小鼠Chr 7，Casr-rs3定位于小鼠Chr 4。我们能够使用特异性引物通过PCR区分Casr-rs1和Casr-rs2，这表明它们是聚集在Chr 7上的不同基因。通过RT-PCR，我们在小鼠肾脏、大脑、睾丸、胚胎和MC3T3-E1成骨细胞中鉴定出了其他Casr-rs转录本，但在肺或肝脏中未检测到。在小鼠肾脏、胚胎和MC3T3-E1成骨细胞中的同源序列，命名为Casr-rs4，其推导氨基酸序列与Casr-rs1的相似度为100%，一致性为97%。从小鼠大脑中扩增出的序列Casr-rs5，其推导蛋白序列与Casr-rs1的相似度为96%，一致性为92%。我们的研究结果证实了小鼠中存在一个新的与Casr相关序列的多成员家族，它们可能编码转导对多种细胞外阳离子反应的受体。
