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Molecular thermodynamics of receptor competition for endocytic uptake.

ABSTRACT: Endocytic uptake of receptors from the cell surface plays an important role in diverse processes from cell signaling to nutrient internalization. Understanding the mechanisms by which endocytic structures select receptors for internalization is of fundamental importance to our understanding of cellular physiology. Binding of receptors to the endocytic protein machinery is known to facilitate receptor loading into endocytic structures. However, many receptor species use the same small set of biochemical motifs to interact with the endocytic machinery, suggesting that receptors may compete for a limited number of binding sites within endocytic structures. Previous studies have shown that such competition can substantially modify receptor uptake. However, a predictive biophysical understanding of this phenomenon is currently lacking. Toward addressing this gap, here we employ quantitative imaging and statistical thermodynamics to measure and predict the competition between two distinct receptor species that are internalized simultaneously from the cell surface. Our studies demonstrate that when receptors compete for the same interactions with the endocytic machinery, their uptake is fundamentally coupled. Importantly, we find that these trends can be quantitatively predicted by a simple thermodynamic analysis. These results suggest that multiple receptor species reach an equilibrium partitioning between endocytic structures and the surrounding plasma membrane as the receptors compete for occupancy within dynamic endocytic structures. More broadly, this work provides a quantitative framework for predicting the impact of competition on receptor uptake, an effect which has the potential to physically couple signaling pathways that impact diverse aspects of cellular physiology.

SUBMITTER: DeGroot ACM

PROVIDER: S-EPMC6918483 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Molecular thermodynamics of receptor competition for endocytic uptake.

DeGroot Andre C M ACM Zhao Chi C LaMonica Megan F MF Hayden Carl C CC Stachowiak Jeanne C JC

Soft matter 20190901 37

Endocytic uptake of receptors from the cell surface plays an important role in diverse processes from cell signaling to nutrient internalization. Understanding the mechanisms by which endocytic structures select receptors for internalization is of fundamental importance to our understanding of cellular physiology. Binding of receptors to the endocytic protein machinery is known to facilitate receptor loading into endocytic structures. However, many receptor species use the same small set of bioc ...[more]

PMID: 31502617

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Project description:SLAM family receptors regulate activation and inhibition in immunity through recruitment of activating and inhibitory SH2 domain containing proteins to immunoreceptor tyrosine based switch motifs (ITSMs). Binding of the adaptors, SAP and EAT-2 to ITSMs in the cytoplasmic regions of SLAM family receptors is important for activation. We analysed the fine specificity of SLAM family receptor phosphorylated ITSMs and the conserved tyrosine motif in EAT-2 for SH2 domain containing signalling proteins. Consistent with the literature describing dependence of CRACC (SLAMF7) on EAT-2, CRACC bound EAT-2 (KD = 0.003 μM) with approximately 2 orders of magnitude greater affinity than SAP (KD = 0.44 μM). RNA interference in cytotoxicity assays in NK92 cells showed dependence of CRACC on SAP in addition to EAT-2, indicating selectivity of SAP and EAT-2 may depend on the relative concentrations of the two adaptors. The concentration of SAP was four fold higher than EAT-2 in NK92 cells. Compared with SAP, the significance of EAT-2 recruitment and its downstream effectors are not well characterised. We identified PLCγ1 and PLCγ2 as principal binding partners for the EAT-2 tail. Both PLCγ1 and PLCγ2 are functionally important for cytotoxicity in NK92 cells through CD244 (SLAMF4), NTB-A (SLAMF6) and CRACC. Comparison of the specificity of SH2 domains from activating and inhibitory signalling mediators revealed a hierarchy of affinities for CD244 (SLAMF4) ITSMs. While binding of phosphatase SH2 domains to individual ITSMs of CD244 was weak compared with SAP or EAT-2, binding of tandem SH2 domains of SHP-2 to longer peptides containing tandem phosphorylated ITSMs in human CD244 increased the affinity ten fold. The concentration of the tyrosine phosphatase, SHP-2 was in the order of a magnitude higher than the adaptors, SAP and EAT-2. These data demonstrate a mechanism for direct recruitment of phosphatases in inhibitory signalling by ITSMs, while explaining competitive dominance of SAP and EAT-2.

Dataset Information

Molecular thermodynamics of receptor competition for endocytic uptake.

Publications

Molecular thermodynamics of receptor competition for endocytic uptake.

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