Identification of peptide and protein ligands for the caveolin-scaffolding domain - Implications for the interaction of caveolin with caveolae-associated proteins
Identification of peptide and protein ligands for the caveolin-scaffolding domain - Implications for the interaction of caveolin with caveolae-associated proteins
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DOI：
10.1074/jbc.272.10.6525
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发表时间：
1997-03-07
影响因子：
4.8
通讯作者：
Lisanti, MP
中科院分区：
文献类型：
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作者：
Couet, J;Li, SW;Lisanti, MP
Caveolin 是一种 21-24-kDa 的整合膜蛋白，是小窝膜的主要成分。我们认为小窝蛋白可作为支架蛋白来组织和浓缩小窝膜内的某些小窝蛋白相互作用蛋白。在这方面，小窝蛋白与多种脂质修饰的信号分子共同纯化，包括 G 蛋白、Src 样激酶、Ha-Ras 和 eNOS，使用几种独立的方法，已表明小窝蛋白的胞质氨基末端结构域的 20 个氨基酸的膜近端区域足以介导这些相互作用，例如，该结构域与 G 蛋白 cu 亚基和 Src 样激酶相互作用，并且可以在功能上抑制它们的活性，这 Caveolin衍生的蛋白质结构域被称为caveolin支架结构域，然而，caveolin支架结构域如何识别这些分子仍然未知。在这里，我们使用caveolin支架结构域作为受体从噬菌体展示库中选择随机肽配体，这些caveolin选择的肽配体富含芳香族氨基酸并具有 在许多情况下，由于特征间隔，一种已知的小窝蛋白相互作用蛋白 G(i2 α) 被用作配体，以进一步研究这种相互作用的性质。 G(i2 α) 和其他 G 蛋白 α 亚基包含一个通常类似于噬菌体展示序列的单个区域。我们表明，源自 G(i2 α) 的短肽序列直接与小窝蛋白支架结构域相互作用，并竞争性抑制小窝蛋白支架结构域与 G(i2 α) 适当区域的相互作用。这种相互作用严格依赖于肽配体内芳香族残基的存在，因为用丙氨酸或甘氨酸替换这些残基可防止它们与小窝蛋白支架结构域相互作用。此外，我们使用这种相互作用来定义小窝蛋白支架结构域内的哪些残基对于识别这些肽和蛋白质配体至关重要。此外，我们发现支架结构域 Caveolins 1 和 3 的结构域均识别相同的肽配体，而 Caveolin-2 内的相应结构域在相同条件下无法识别这些配体。这些结果有助于进一步证明这种相互作用的特异性。我们目前的研究结果的含义是关于其他 Caveolin 和 Caveolae 相关蛋白的讨论。
Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes, We have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting proteins within caveolae membranes, In this regard, caveolin co purifies with a variety of lipid-modified signaling molecules, including G-proteins, Src-like kinases, Ha-Ras, and eNOS, Using several independent approaches, it has been shown that a 20-amino acid membrane proximal region of the cytosolic amino-terminal domain of caveolin is sufficient to mediate these interactions, For example, this domain interacts with G-protein cu subunits and Src-like kinases and can functionally suppress their activity, This caveolin-derived protein domain has been termed the caveolin scaffolding domain, However, it remains unknown how the caveolin-scaffolding domain recognizes these molecules.Here, we have used the caveolin-scaffolding domain as a receptor to select random peptide ligands from phage display libraries, These caveolin selected peptide ligands are rich in aromatic amino acids and have a characteristic spacing in many cases, A known caveolin-interacting protein, G(i2 alpha), was used as a ligand to further investigate the nature of this interaction. G(i2 alpha), and other G-protein alpha subunits contain a single region that generally resembles the sequences derived from phage display. We show that this short peptide sequence derived from G(i2 alpha) interacts directly with the caveolin-scaffolding domain and competitively inhibits the interaction of the caveolin-scaffolding domain with the appropriate region of G(i2 alpha). This interaction is strictly dependent on the presence of aromatic residues within the peptide ligand, as replacement of these residues with alanine or glycine prevents their interaction with the caveolin-scaffolding domain, In addition, we have used this interaction to define which residues within the caveolin-scaffolding domain are critical for recognizing these peptide and protein ligands, Also, we find that the scaffolding domains of caveolins 1 and 3 both recognize the same peptide ligands, whereas the corresponding domain within caveolin-2 fails to recognize these ligands under the same conditions, These results serve to further demonstrate the specificity of this interaction, The implications of our current findings are discussed regarding other caveolin- and caveolae-associated proteins.