RESUMO
The medium chain mu 2 subunit (AP50) of the clathrin-associated adapter protein complex 2 (AP-2) interacts specifically with the tyrosine-based signals of several integral membrane proteins through the consensus sequence YXXPhi, where X can be any residue and Phi is a large hydrophobic residue. Using surface plasmon resonance combined with structural information, we have analysed the interaction of AP50 with peptides derived from the cytoplasmic tail of cytotoxic T-lymphocyte antigen 4 (CTLA-4). The crystal structure of AP50 in complex with a CTLA-4-derived peptide was determined to 3.6 A (1 A=0.1 nm) resolution. The binding domain of AP50 (residues 164-435) was expressed in Escherichia coli and purified. In agreement with previous reports, the AP50 domain bound to residues 152-174 of CTLA-4, but not to the same peptide that was phosphorylated at the single tyrosine residue (position 165). The interaction exhibited fast kinetics with rapid on and off rates and a K(d) of 0.7 microM. In order to further understand why AP50 binds to CTLA-4, but not to the homologous receptor CD28, a comparison of binding of AP50 with five peptides with single changes in and around the YXXPhi motif to the equivalent residues of CD28 was made. T162H greatly reduced binding, whereas T161L had little effect. Mutations G163S, V164D and K167N all exhibited reduced binding. Modelling of the single amino acid changes using structural information, was in broad agreement with the binding data, demonstrating that residues outside of the YXXPhi motif are also important in the interaction of membrane proteins with AP50.
Assuntos
Complexo 2 de Proteínas Adaptadoras , Subunidades mu do Complexo de Proteínas Adaptadoras , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Clatrina/metabolismo , Imunoconjugados , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Abatacepte , Proteínas Adaptadoras de Transporte Vesicular , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Antígenos CD , Antígenos de Diferenciação/química , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Sequência de Bases , Sítios de Ligação , Antígenos CD28/química , Antígenos CD28/genética , Antígenos CD28/metabolismo , Antígeno CTLA-4 , Proteínas de Transporte/genética , Cristalografia por Raios X , Primers do DNA/genética , Humanos , Técnicas In Vitro , Cinética , Substâncias Macromoleculares , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
We have introduced a Ren-1D targeting vector into embryonic stem cells containing the two highly homologous mouse renin genes Ren-1D and Ren-2. Using a polymerase chain reaction (PCR) screen designed to detect targeted integration at Ren-1D and Ren-2, we isolated 15 targeted embryonic stem cell clones, all of which had undergone a gene conversion event at the Ren-1D locus. We did not isolate any clones in which the incoming DNA had recombined with Ren-2. Over the region encompassed by our transgene, Ren-1D and Ren-2 display greater than 95% homology. Our results suggest that the machinery driving gene targeting by means of homologous recombination in mammalian cells is capable of distinguishing between these two sequences. Construction of transgenic mice with the embryonic stem cells reported here carrying a mutated renin gene will permit a greater understanding of the functions of the Ren-1D and Ren-2 gene products and their relative contribution to cardiovascular homeostasis.