Receptor-MediateReceptor-Mediated Endocytosisd Endocytosis

受体介导的受体介导的内吞作用内吞作用

组员：组员：康艳春 张柳康艳春 张柳

IntroductionIntroduction Receptor mediated endocytosis(RME) is an en

docytotic mechanism in which specific molecules are ingested into the cell. The specificity results from a receptor-ligand interaction. Receptors on the plasma membrane of the target tissue will specifically bind to ligands

on the outside of the cell. An endocytotic process occurs and the ligand is ingested.

In 1964,Thomas Roth and Keith Porter reported on the mechanism by which yolk proteins might be taken into the oocytes （卵母细胞） of mosquitoes

报道了蚊子的卵黄蛋白进入卵母细胞的可能机制

一项实验研究：提出问题→观察→推测

Raise the question: How are the high-molecular-weight yolk proteins able to enter the oocyte?

问题的提出： 高相对分子质量的卵黄蛋白如何进入卵黄细胞？

Notice: during stages of rapid oocyte growth, the

re was a dramatic increase in the number of pitlike depressions seen on the surface of the oocyte.

注意到：在卵黄细胞快速生长的阶段，其细胞表面小窝样凹陷的数目剧增，这些小窝是由质膜的内陷形成的，并且在小窝的内表面覆盖着一层刚毛状的外被。

Visualization of coated pits (P) and vesicles (V) suggested that endocytosis was specific. ROTH

Postulation: yolk proteins were specifically adsorbed onto the outer surface of the membrane of the coated pits , which would then invaginate as coated vesicles.

推测：卵黄蛋白特异地吸附在有被小窝膜的外表面，然后内陷为包被小泡。

这些小泡失去它们的刚毛状的外被，相互融合形成大的膜包被的卵黄体，这是成熟的卵黄细胞的特征。

The first insight into the structThe first insight into the structure of coated vescilesure of coated vesciles

Electron microscopic examination: a crude vesicle fraction isolated from guinea pig brains （Figure1）

从豚鼠脑髓分离出粗制的小泡组分进行电镜观察

Show: coated vesicles were covered by a polygonal basketwork.

表明：包被小泡表面覆盖着多边形的编织物

Suggestion: the coatings were an apparatus to control the infolding of the plasma membrane during vesicle formation.

研究者提出：外被是在小泡形成过程中用来控制质膜折叠的装置

The first studies of the The first studies of the biochemical nature of the biochemical nature of the

vesicle coatvesicle coat

方法：将来自猪脑的小泡经过连续的蔗糖密度梯度离心，直到获得纯化的包被小泡。包被小泡的蛋白质被溶解后经过 SDS- 聚丙烯酰胺凝胶电泳分离。

结果表明：外被含一种相对分子质量约为180000 的优势蛋白质。 Pearse 将其命名为网格蛋白

普遍性 : 他再从其他几种动物的不同类型的细胞中分离的包被小泡的样品中，基于相对分子质量和肽谱分析也发现了同样的蛋白质

Brown and Goldstein had Brown and Goldstein had become Interested in the become Interested in the

inherited condition inherited condition familial familial hypercholesterolemia (FH)hypercholesterolemia (FH)

People who were homozygous ( 纯合子 )for th

e defective gene had profoundly elevated levels of serum cholesterol (800mg/dl vs.200mg/dl for a normal person), invariably developed severely blocked arteries (atherosclerotic 动脉粥样硬化 ) and usually died from heart attack before the age of 20. At the time, very little was known about the fundamental physiologic or biochemical defects in this disorder.

familial hypercholesterolemifamilial hypercholesterolemia (FH)a (FH) 对缺陷基因 (FH 等位基因 ) 为纯合子的人，

血清胆固醇的水平明显升高达 800mg/dL（正常水平为 200mg/dL ），全部产生严重的动脉阻塞（动脉粥状硬化），通常在 20 岁前死于心脏病发作。那时对这种病症的基本生理或生化缺陷知之甚少。

What is FH?

What Causes FH?

How is FH Inherited?

What is FH?What is FH?• Familial Hypercholesterolemia (FH) is an inherited

disorder that causes very high cholesterol levels and greatly increases the chance of having a heart attack early in life.  Heart attacks usually occur in men when they are 40-55 years old and in women when they are 50-65 years old.  Unfortunately, they can sometimes occur when people are in their mid-twenties.  The first case of FH was described over 100 years ago and the characteristics of FH passing from generation to generation was understood in the late 1930's.  Actual genetic proof was discovered in the 1960's and 1970's.

What Causes FH?What Causes FH?

Cholesterol is removed from the blood by the liver using Low Density Lipoprotein (LDL) receptors. Each person has two genes that are responsible for making the LDL receptors: one received from the father and one received from the mother.  In a person with FH, an abnormal gene was passed on from one parent who has FH and a normal gene was passed on from the other parent. Therefore, half of the LDL receptors are absent or do not work properly and the other half are normal. Because half of the receptors do not remove the cholesterol normally, cholesterol levels increase in the blood. This  results in damage to blood vessels, blockage of arteries and heart attacks at an early age.

How is FH Inherited?How is FH Inherited?

If a person has FH, then each of his or her children will have a 50% chance of inheriting FH.  You are either born with FH or not.  Cholesterol levels can be checked at birth, but the National Cholesterol Education Program (NCEP) guidelines suggest children in a FH family be checked at age two. Most persons with the disease are neither recognized nor treated.

their studies of FH their studies of FH 方法：他们从正常个体和 FH 受累个体皮肤

分离出成纤维细胞进行培养，观察胆固醇代谢。

发现：在正常的成纤维细胞中，胆固醇生物合成的速率控制酶（ rate-controlling enzyme),即 HMGCoA还原酶，它们的活性被含有胆固醇的脂蛋白（如 LDL ）所抑制。（如图所示）

所以，在正常成纤维细胞生长的培养基中加入所以，在正常成纤维细胞生长的培养基中加入 LDLLDL导致导致ＨＭＧＣｏＡＨＭＧＣｏＡ还原酶活性的降低，相应成纤维还原酶活性的降低，相应成纤维

细胞中胆固醇的合成水平也降低细胞中胆固醇的合成水平也降低

测量正常成纤维细胞中的HMG CoA 还原酶活性。空心方块：加入牛血清的脂蛋白组分

实心圈：未分级分离的牛血清

空心三角：牛血清的非脂蛋白组分

从图中明显看出脂蛋白可以降低酶的活性，而非脂蛋白对酶活性几乎没有影响。

• 当检测 FH 衍生的成纤维细胞中 HMG ＣｏA 还原酶水平时，发现它们是正常成纤维细胞的 40~60 倍。另外，全部 FH 成纤维细胞中的酶活性不受在环境媒介中 LDL 出现的影响。（图 3 ）

• 在第六天（ 0h ）时，培养基被新鲜的培养基替代。其中含有 5% 人类缺脂蛋白的人的血浆的培养基

• 正常的对照组细胞（实心圈）开始具有很低的酶活性，因为培养基中有足够的含胆固醇的脂蛋白，细胞不必自己合成，一旦培养基换成缺失胆固醇的血浆，细胞不能再利用培养基中的胆固醇，于是细胞内的酶量增加。相比之下，来自 FH患者的细胞（空心圈）对培养基中脂蛋白的存在与否没有反应

Studies on the interaction Studies on the interaction between the cells and the between the cells and the

lipoproteinslipoproteins How could lipoproteins in the medium affe

ct the activity of an enzyme in the cytoplasm of cultured cells? 　　

环境中的脂蛋白是怎样影响人工培养细胞的细胞质中的酶活性呢？

• 为了解释这个问题， Brown和 Goldstein开始了对细胞和脂蛋白之间相互影响的研究。

• To answer this question, Brown and Goldstein initiated studies on the interaction between the cells and the lipoproteins.

Method:They added radioactively labeled LDL to culture dishes containing a single layer of fibroblasts derived from either FH-afflicted or normal human subjects.

方法：他们将放射性标记的 LDL加入含有一层成纤维细胞的培养皿中，这些成纤维细胞来自 FH 受累个体或正常人。

• 圆形表示放射性 [125I ] 标记的 LDL 在 37℃与正常的细胞结合。三角形表示与 FH 的细胞结合。

• 空心圆和三角为细胞在含有 5mg/ml[125I]LDL缓冲液的 250mg/ml非放射性 LDL 中生长。实心圆和三角则不含有非放射性 LDL 。

• 在没有添加非放射性 LDL 的细胞中很明显看出，正常细胞结合了一定数量的 LDL ，而 FH患者细胞则不能结合。

• 而在添加了非放射性 LDL 的细胞中标记 LDL 大量减少。因为非标记脂蛋白与标记的 LDL竞争结合。

we can see from the figure that in the absence of added nonradioactivity LDL,the normal cells bind to the labeled LDL significantly,while the cells from the FH patients do not. The binding of the labeled LDL is greatly reduced in the presence of nonradioactivity LDL,because nonlabeled lipoprotein compete with the labeled ones for binding sites .

ConclusionConclusion

　　这些结论表示出正常细胞有一个 LDL 的高特异性的受体，这个受体在有缺陷的或者在 FH病人的细胞中丢失。

　　 These results indicated that normal cells have a highly specific receptor for LDL and that this receptor was defective or missing in cells from patients with FH.

To discover the process of receptor binding and internalization(为了观察受体结合和内化作用的过程 ),some scientists did other researches.

Now, let’s welcome kangyanchun to go on talking about them.