RESUMO
The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the trans-Golgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC).
Assuntos
Antígenos CD/metabolismo , Selectina-P/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Corpos de Weibel-Palade/metabolismo , Complexo 3 de Proteínas Adaptadoras , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Humanos , Migração e Rolagem de Leucócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Eletrônica , Modelos Biológicos , Selectina-P/química , Selectina-P/genética , Sinais Direcionadores de Proteínas/genética , Estrutura Terciária de Proteína , Transporte Proteico , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tetraspanina 30 , Fatores de Transcrição/metabolismo , Corpos de Weibel-Palade/ultraestrutura , Rede trans-Golgi/metabolismoRESUMO
Weibel-Palade bodies (WPBs) are the lysosome-related secretory organelles of endothelial cells. Their content protein von Willebrand factor, plays a key role in haemostasis, whilst P-selectin in the membranes is critical in the initiation of inflammation. Biogenesis of these rod-shaped structures is driven by von Willebrand factor, since its heterologous expression leads to formation of organelles morphologically indistinguishable from bona fide WPBs. The two main membrane proteins of WPBs, CD63 and P-selectin, have complex itineraries controlled largely by cytoplasmic targeting signals. We are only just beginning to understand the way in which these three proteins come together to form mature WPBs.
Assuntos
Corpos de Weibel-Palade/fisiologia , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Humanos , Dados de Sequência Molecular , Selectina-P/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Tetraspanina 30RESUMO
The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.
