Expression and purification of the extracellular ligand-binding domain of the atrial natriuretic peptide (ANP) receptor: monovalent binding with ANP induces 2:2 complexes.
Expression and purification of the extracellular ligand-binding domain of the atrial natriuretic peptide (ANP) receptor: monovalent binding with ANP induces 2:2 complexes.
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心房钠尿肽 (ANP) 受体胞外配体结合域的表达和纯化：与 ANP 的单价结合诱导 2:2 复合物。
DOI：
10.1021/bi982127v
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Zhang,X
中科院分区：
文献类型：
--
作者：
Misono,KS;Sivasubramanian,N;Berkner,K;Zhang,X
心房利钠肽（ANP）受体是一种I型跨膜蛋白，其含有胞外配体结合结构域、单一跨膜序列、胞内激酶同源结构域和鸟苷酸环化酶（GCase）结构域。ANP与细胞外结构域的结合导致GCase结构域的激活，其机制尚不清楚。为了便于受体结构和信号传导机制的研究，我们以水溶性形式表达了大鼠ANP受体的胞外ANP结合结构域（NPR-ECD）。通过ANP-亲和层析将NPR-ECD纯化至均一。SDS−PAGE显示单一的61-kDa条带，与用N4 α-叠氮苯甲酰基-125 I-ANP（4−28）光亲和标记获得的放射性条带一致。Edman降解得到了成熟蛋白预期的单个氨基末端序列。三氟甲磺酸和肽-N-糖苷酶F处理均产生50 kDa条带，表明N-糖基化。通过质谱法测定的57 725 Da的分子量表明碳水化合物含量为16%。NPR-ECD以与全长受体相当的亲和力结合ANP。NPR-ECD的配体选择性（顺序为ANP >脑钠肽> NPR-ECD-型钠肽）也与全长受体相似。NPR-ECD的HPLC凝胶过滤得到表观质量为74 kDa的峰。与ANP预孵育产生了一个新的150-kDa的峰，伴随着74-kDa的峰减少。峰位置的这种偏移是ANP浓度依赖性的，并且在NPR-ECD与ANP摩尔比为1：1时完全，表明等摩尔结合。表观天然分子量从74 kDa变为150 kDa，表明结合导致NPR-ECD：ANP复合物二聚化，产生[NPR-ECD：ANP] 2复合物。
The receptor for atrial natriuretic peptide (ANP) is a type-I transmembrane protein containing an extracellular ligand-binding domain, a single transmembrane sequence, an intracellular kinase-homologous domain, and a guanylate cyclase (GCase) domain. Binding of ANP to the extracellular domain causes activation of the GCase domain by an as yet unknown mechanism. To facilitate studies of the receptor structure and signaling mechanism, we have expressed the extracellular ANP-binding domain of rat ANP receptor (NPR-ECD) in a water-soluble form. NPR-ECD was purified to homogeneity by ANP-affinity chromatography. SDS−PAGE gave a single 61-kDa band, which coincided with a radioactive band obtained by photoaffinity-labeling withN4α-azidobenzoyl-125I-ANP(4−28). Edman degradation gave a single amino-terminal sequence expected for the mature protein. Both trifluoromethanesulfonic acid and peptide-N-glycosidase F treatments yielded a 50-kDa band, indicatingN-glycosylation. The molecular mass of 57 725 Da determined by mass spectrometry indicates the carbohydrate content at 16%. NPR-ECD bound ANP with an affinity comparable to that of the full-length receptor. The ligand selectivity of NPR-ECD (in the order ANP > brain natriuretic peptide ≫ C-type natriuretic peptide) was also similar to that of the full-length receptor. HPLC gel filtration of NPR-ECD gave a peak with an apparent mass of 74 kDa. Preincubation with ANP generated a new 150-kDa peak with a concomitant decrease of the 74-kDa peak. This shift in peak positions was ANP concentration-dependent and was complete at the NPR-ECD-to-ANP molar ratio of 1:1, indicating equimolar binding. The change in the apparent native molecular weight from 74 to 150 kDa suggests that binding causes dimerization of the NPR-ECD:ANP complex to yield an [NPR-ECD:ANP]2complex.