Leveraging a Novel ITIM Motif in GPCRs for Targeted Antibody Design
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CXCR4, a chemokine GPCR, is essential for migration of neuronal, hematopoietic, and breast cancer cells during metastasis whereby CXCR4 dysregulation promotes migration and invasion. Following SDF stimulation, CXCR4 is phosphorylated on Ser/Thr residues which initiates adaptor recruitment, receptor desensitization, and trafficking to endocytic sites. Here we show that stimulation with gradient SDF, delays receptor phosphorylation and trafficking, leading to sustained signaling to a novel CXCR4‐SHP2‐ERK pathway. SHP2 is a tyrosine phosphatase implicated in HER2(+) and triple‐negative breast cancers, whereby it transduces mitogenic and migratory signals driving hyperproliferation and invasion. SHP2 is recruited to tyrosine phosphorylated ITIM motifs ( i mmmunoreceptor t yrosine‐based i nhibitory consensus m otifs), a hallmark found in inhibitory immune receptors with little evidence in GPCRs. Here we identify an ITIM motif in CXCR4 that regulates both SHP2 binding and signaling. Specifically, we assessed if gradient SDF stimulation of CXCR4 1) delays receptor phosphorylation and trafficking, 2) sustains signaling to SHP2‐ERK, 3) induces SHP2‐dependent migration; and if CXCR4 Tyr mutation within the ITIM motif 4) maintains SDF gradient sensing ability, and 5) disrupts interaction with and signaling to SHP2. Our data demonstrate that gradient SDF delays receptor Ser/Thr phosphorylation and internalization thereby sustaining signaling to SHP2‐ERK and driving SHP2‐dependent migration. Furthermore, the ITIM mutant maintains SDF gradient sensing ability, but disrupts interaction with and signaling to SHP2. Our data support a working model that CXCR4 contains a functional ITIM motif which we are currently leveraging for targeted antibody design for use in migration studies of aggressive breast cancer cells with dysregulated CXCR4. Support or Funding Information These studies were supported by NIH grant GM‐097718, PA Department of Health grant SAP4100057688, and the Milton Lev Memorial Faculty Research Fund.Keywords:
Internalization
Endocytosis of plasma membrane proteins is mediated by their interaction with adaptor proteins. Conversely, emerging evidence suggests that adaptor protein recruitment to the plasma membrane may depend on binding to endocytic cargo. To test this idea, we analyzed the yeast adaptor protein Sla1, which binds membrane proteins harboring the endocytic signal NPFxD via the Sla1 SHD1 domain. Consistently, SHD1 domain point mutations that disrupted NPFxD binding caused a proportional reduction in Sla1-GFP recruitment to endocytic sites. Furthermore, simultaneous SHD1 domain point mutation and deletion of the C-terminal LxxQxTG repeat (SR) region linking Sla1 to coat proteins Pan1 and End3 resulted in total loss of Sla1-GFP recruitment to the plasma membrane. These data suggest that multiple interactions are needed for recruitment of Sla1 to the membrane. Interestingly, a Sla1 fragment containing just the third SH3 domain, which binds ubiquitin, and the SHD1 domain displayed broad surface localization, suggesting plasma membrane recruitment is mediated by interaction with both NPFxD-containing and ubiquitylated plasma membrane proteins. Our results also imply that a Sla1 NPF motif adjacent to the SR region might regulate the Sla1-cargo interaction, mechanistically linking Sla1 cargo binding to endocytic site recruitment.
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Signaling is regulated by endocytosis at multiple levels along endocytic routes. Endocytic control of signaling starts already at the plasma membrane, where cells employ different mechanisms to finely tune the type and strength of signals emanating from the cell surface. Here, we will review some of the most recently described endocytic mechanisms controlling signaling at the plasma membrane, through the regulation of internalization dynamics and through the integration of different internalization pathways triggered by canonical chemical stimuli or physical forces.
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Cells need to exchange material and information with their environment. This is largely achieved via cell-surface receptors which mediate processes ranging from nutrient uptake to signaling responses. Consequently, their surface levels have to be dynamically controlled. Endocytosis constitutes a powerful mechanism to regulate the surface proteome and to recycle vesicular transmembrane proteins that strand at the plasma membrane after exocytosis. For efficient internalization, the cargo proteins need to be linked to the endocytic machinery via adaptor proteins such as the heterotetrameric endocytic adaptor complex AP-2 and a variety of mostly monomeric endocytic adaptors. In line with the importance of endocytosis for nutrient uptake, cell signaling and neurotransmission, animal models and human mutations have revealed that defects in these adaptors are associated with several diseases ranging from metabolic disorders to encephalopathies. This review will discuss the physiological functions of the so far known adaptor proteins and will provide a comprehensive overview of their links to human diseases.
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The putative seven-transmembrane (TM) domains have been the structural hallmark for the superfamily of heterotrimeric G protein-coupled receptors (GPCRs) that regulate a variety of cellular functions by mediating a large number of extracellular signals. Five-TM GPCR mutants of chemokine receptor CCR5 and CXCR4, the N-terminal segment of which connected directly to TM3 as a result of a deletion of TM1–2 and the first intracellular and extracellular loops, have been obtained in this study. Laser confocal microscopy and flow cytometry analysis revealed that these five-TM mutant GPCRs were expressed stably on the cell surface after transfection into human embryonic kidney 293 cells. The five-TM CCR5 and CXCR4 functioned as normal chemokine receptors in mediating chemokine-stimulated chemotaxis, Ca 2+ influx, and activation of pertussis toxin-sensitive G proteins. Like the wild-type GPCRs, the five-TM mutant receptors also underwent agonist-dependent internalization and desensitization and were subjected to regulation by GPCR kinases and arrestins. Our study indicates that five-TM domains, at least in the case of CCR5 and CXCR4, appear to meet the minimum structural requirements for a functional GPCR and suggests possible existence of functional five-TM GPCRs in nature during evolution.
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Endocytosis effectuates a critical interface between the eukaryotic cell and its apposing environment. It is, subsequently, paramount for many physiologically important processes and encompasses a diverse array of mechanisms and pathways. The classical endocytic routes mediated by clathrin and caveolin are the best understood and the molecular roles of their major regulators, such as dynamin, adaptor proteins and various lipid species, are the most comprehensively described. Recent identification of an assortment of constitutive, noncaveolar, clathrin-independent endocytic (CIE) pathways has expanded the endocytic system. Unlike the classical endocytic pathways, little is known about the guiding parameters of CIE routes. Consequently, it is not possible to understand the important cellular roles these pathways may be fulfilling. This study has begun to characterise the very basic parameters governing the morphologically striking Clathrin-Independent Carrier (CLIC) pathway. Development of a diverse molecular toolkit has now allowed the quantitation of endocytic capacity provided by CLICs, the visualisation of subtle sorting components of the CLIC pathway, the isolation of novel CLIC cargo and regulators, and has linked this mechanism to the critical cellular processes of cellular migration and membrane repair. Calculation of the individual capacity of endocytic routes provides important information about the contribution of each pathway to total plasma membrane (PM) uptake and turnover. Quantitation of the volume, surface area and number of structures forming per minute in this study shows that CLICs provide the vast majority of constitutive endocytosis, up to four times the capacity of the clathrin mediated endocytic (CME) pathway. As the equivalent of the entire PM area could pass through the CLIC pathway within 12 minutes it is evident that CLICs are fundamental housekeepers of bulk membrane internalisation. Thus, they are likely to be central regulators of PM homeostasis and turnover. High-resolution tomography, in conjunction with analysis of CLIC cargo trafficking, identifies these carriers as complex, pleiomorphic structures that sort the bulk of membrane to early endosomes and recycle cargo back to the cell surface. Such vast internalisation combined with an ability to rapidly recycle components quickly attributes the CLIC pathway as a complex sorting station. Isolation of novel cargo and regulators has identified a striking array of proteins now associated with the CLIC pathway for the first time. A significant proportion of identified targets localise to lipid-rafts and recycle from the PM, facets consistent with association to the CLIC pathway. Numerous targets have also been directly implicated in clathrin-independent endocytosis by independent groups. Verification of selected cargo, such as CD44, Thy-1 and myoferlin, showing specific internalisation through the CLIC pathway, has provided insight into the sorting ability of the CLIC pathway and links to adhesion turnover and membrane recycling. Consistent with a role in cellular adhesion turnover, it was found that CLICs become polarised within migrating cells. This has shown the first instance of spatial separation between three major endocytic routes, CLICs, caveolae and CME and highlights the important and coordinated roles of multiple endocytic pathways during physiologically significant processes. The specific internalisation of paxillin, Thy-1 and CD44 through CLICs at the leading edge of migrating cells suggests that CLICs rapidly turnover adhesion components for dynamic extracellular sensation during directional cell migration. Indeed, specific ablation of the CLIC pathway significantly impedes cellular migration, implying coordination with CME at the leading edge. This study has defined numerous parameters of the CLIC pathway, developing the current understanding of this poorly defined route and places the CLIC pathway as a unique player during critical cellular processes.
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尽管花粉试管生长是为更高的植物授精和种子生产的一个前提，导致花粉试管排放和延伸的过程为理解尖端生长的基本机制是关键的。在他们与顶端的血浆膜(下午) 的一个限制区域被认为到保险丝的地方，花粉试管延伸在顶端的区域，或清楚的地区由导出 Golgi 的能分泌的泡(SV ) 的累积和熔化发生，这通常被接受，定义顶端的生长领域。在尖端的 SV 的熔化在房间墙材料外面逆行并且提供下午的新片断。然而，电子显微镜学研究清楚地证明了极大地在尖端合并的 PM 超过延伸，下午检索的机制已经被要求在中间 -- 第十九个世纪。endocytosis 上的最近的研究在花粉试管生长期间证明不同 endocytic 小径发生在试管的不同地区，包括顶，并且导致了一个新假设在尖端解释泡累积；也就是， endocytic 泡除了 SV 和那 exocytosis 实质地作出贡献到塑造 V 的泡累积，这不包含全部顶端的领域。新卓见建议了在在顶的 exo- 和 endocytosis 之间的调整贡献的吸引人的假设以类脂化合物 / 蛋白质作文和能在房间的生理学有不同功能的显示出的不同降级小径维持下午极性。在 vivo 的花粉试管生长被和风格分子的相互作用仔细调整。在花粉试管再循环的 endocytosis 和膜的学习打开新观点到在 vivo 学习花粉试管风格相互作用。
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Casein kinase 2
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Endocytic Transport and Some of its Implications for Physiology, Metabolism, and Disease From a systems biology perspective, endocytic transport can be considered as cell subsystem whose function has major implications for homeostasis, as well as for basic responses of a cell in terms of survival, proliferation, differentiation, and generation of external signals (intercellular communication). As such, this subsystem has implications for many physiological and pathological processes (examples below). At least eight or nine different endocytic pathways have been characterized or postulated. Several of these can participate in Receptor-Mediated Endocytosis (RME).
Cell Physiology
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