Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein.
Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein.
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肝质膜上的血管活性肠肽受体：作为糖蛋白的表征。
DOI：
10.1021/bi00350a013
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Gray,GM
中科院分区：
文献类型：
--
作者：
Nguyen,TD;Williams,JA;Gray,GM
斯坦福大学医学院消化内科，斯坦福大学，加州94305，和加州大学生理学和医学系，弗朗西斯科，加州94143接收日期：1985年7月23日摘要：用~（125）I-共价交联法在大鼠肝细胞膜上发现血管活性肠肽（VIP）受体。VIP通过三种不同的试剂[二琥珀酰亚胺二硫代双（丙酸酯），二琥珀酰亚胺辛二酸酯和琥珀酰亚胺4-叠氮基苯甲酸酯]，并通过十二烷基硫酸钠-丙烯酰胺电泳进行检查。无论还原条件的存在下，两种分子种类的推定VIP结合单位被确定为广泛的放射自显影带的80 000和56 000道尔顿（Da）。无论是大的和小的物种表现出相同的高亲和力125 I-VIP结合和随后的交联（在3 nM未标记的VIP的半最大抑制）。80-kDa物质在变性条件下部分转化为56-kDa形式，当与1 pg/mL胰凝乳蛋白酶、胰蛋白酶或弹性蛋白酶在20 ℃孵育30分钟时，被广泛降解为类似于56-kDa单位迁移的片段。相比之下，56-kDa部分对丝氨酸蛋白酶的攻击具有抗性。80-和56-kDa物质均为微异质性，至少部分原因是存在碳水化合物链，每种物质均与麦胚凝集素（WGA）-琼脂糖（~ 50%）部分结合。与WGA未结合的级分相比，WGA结合的级分（用A '-乙酰葡糖胺洗脱）在丙烯酰胺凝胶上相对延迟。将80-和56-kDa物质暴露于内-/3-乙酰氨基葡萄糖苷酶F，使每个物质的表观分子量减少19 kDa，表明存在复杂的N-连接碳水化合物。受体种类似乎没有高甘露糖N-连接链，因为它们不与伴刀豆球蛋白A相互作用，也不被内切-β-乙酰氨基葡萄糖苷酶H切割。125 I-VIP标记的受体很容易用1%Triton X-100溶解，并从Fractogel 55 F中洗脱出200 kDa的单位和47 kDa的非糖基化片段，这是溶解过程的假象。80-和56-kDa的物种的溶解受体的组件。肝VIP受体似乎是一种大的糖蛋白，可能由一个或多个77-kDa单位组成（交联VIP的质量少80 kDa），其中19 kDa由复杂的N-连接的碳水化合物链组成。该VIP结合单元与其自身或与一个或多个修饰亚基非共价复合。一个较小的53-kDa部分，虽然可能是一个真正的伴侣较大的结合亚基，更有可能是一个部分降解形式的受体。肝VIP受体在结构上与胰高血糖素受体、小肠上皮细胞和人淋巴母细胞VIP受体不同，血管活性肠肽（VIP）是一种神经递质和激素，广泛作用于多种组织。与这种普遍存在的作用相对应，VIP受体已在胃肠道（唾液腺、胃平滑肌、胃细胞、肠上皮细胞、胰腺腺泡和胰岛）、中枢神经系统、肺、心脏、血管、子宫平滑肌、肾上腺、视网膜、脂肪细胞和不同类型的淋巴细胞中被鉴定（Said，1984）。在胃肠系统中，VIP作用于平滑肌以影响运动性并诱导肠液和胰腺液以及电解质的分泌。VIP在肝脏中的生理作用尚不确定。虽然早期的证据显示...
Division of Gastroenterology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, and Departments of Physiology and Medicine, University of California, San Francisco, California 94143 Received July 23, 1985 abstract: The receptor for vasoactive intestinal peptide (VIP) was identified inrat liver plasma membranes after covalent cross-linking to 125I-VIP by three different agents [disuccinimido dithiobis (propionate), disuccinimido suberate, and succinimido 4-azidobenzoate] and examined by sodium dodecyl sulfate-acrylamide electrophoresis. Regardless of the presence of reducing conditions, two molecular species of the putative VIP binding unit were identified as broad autoradiographic bands of 80 000 and 56 000 daltons (Da). Both the largeand small species showed the same high affinity for 125I-VIP binding and subsequent cross-linking (half-maximal inhibition at 3 nM unlabeled VIP). The 80-kDa species was partially converted to the 56-kDa form by denaturing conditions and was extensively degraded when incubated at 20 C for 30 min with 1 pg/mL chymotrypsin, trypsin, or elastase tofragments that migrated similarly to the 56-kDa unit. In contrast, the 56-kDa moiety was resistant to attack by serine proteases. Both the 80-and 56-kDa species were microheterogeneous due at least in part to the presence of carbohydrate chains, each species binding fractionally to wheat germ agglutinin (WGA)-agarose (~ 50%). The WGA-bound fraction (eluted with A'-acetylglucosamine) was relatively retarded on acrylamide gels as compared to the WGA-unbound fraction. Exposure of the 80-and56-kDa species toendo-/3-acetylglucosaminidase F reduced the apparent molecular mass of each by 19 kDa, indicating the presence of complex N-linked carbohydratechains. The receptor species do not appearto have high-mannose N-linked chains since they did not interact with concanavalin A and were not cleaved by endo-/3-acetylglucosaminidase H. The125I-VIP-labeled receptor was readily solubilized with 1% Triton X-100 and eluted from Fractogel55F as a 200-kDa unit and a 47-kDa nonglycosylated fragment that was an artifact of the solubilization process. Both the 80-and 56-kDa species were components of the solubilized receptor. The hepatic VIP receptor appears to be a large glycoprotein probably consisting of one or more 77-kDa units (80 kDa less the mass of the cross-linked VIP), 19 kDa of which is accounted for by complex N-linked carbohydrate chains. This VIP binding unit is complexed noncovalently either with itself or with one or more modifying subunits. A smaller 53-kDa moiety, though possibly a true companion to the larger binding subunit, is more likely to be a partially degraded form of the receptor. The hepatic VIP receptor is structurally distinct both from the hepatic glucagon receptor and from the VIP receptors in the intestinal enterocyte and in human lymphoblasts.Vasoactive intestinal polypeptide (VIP), 1 a neurotransmitter and hormone, has widespread actions on many tissues. Commensurate with this ubiquitous role, VIP receptors have been identified in the gastrointestinal tract (salivary gland, gastric smooth muscle, gastric cells, intestinal epithelial cells, pan-creatic acini and islets), the central nervous system, the lung, the heart, the blood vessels, the uterine smooth muscle, the adrenal gland, the retina, the adipocytes, and different types of lymphocytes (Said, 1984). In the gastrointestinal system, VIP acts on smooth muscle to influence motility and induces secretion of intestinal and pancreatic fluid and electrolytes. The physiologic role of VIP in the liver is uncertain. While early evidence suggested a …