Post-translational allosteric activation of the P2X7 receptor through glycosaminoglycan chains of CD44 proteoglycans

Gedd Moura,Ej Paredes-Gamero,Ma Lima,Tf Gesteira,Hb Nader,Ils Tersariol,Fd Nascimento,Sv Lucena

doi:10.1038/cddiscovery.2015.5

Gedd Moura, Ej Paredes-Gamero + Show 6 more

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https://doi.org/10.1038/cddiscovery.2015.5

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Abstract

Here, we present evidence for the positive allosteric modulation of the P2X7 receptor through glycosaminoglycans (GAGs) in CHO (cell line derived from the ovary of the Chinese hamster) cells. The marked potentiation of P2X7 activity through GAGs in the presence of non-saturating agonists concentrations was evident with the endogenous expression of the receptor in CHO cells. The presence of GAGs on the surface of CHO cells greatly increased the sensitivity to adenosine 5′-triphosphate and changed the main P2X7 receptor kinetic parameters EC50, Hill coefficient and Emax. GAGs decreased the allosteric inhibition of P2X7 receptor through Mg2+. GAGs activated P2X7 receptor-mediated cytoplasmic Ca2+ influx and pore formation. Consequently, wild-type CHO-K1 cells were 2.5-fold more sensitive to cell death induced through P2X7 agonists than mutant CHO-745 cells defective in GAGs biosynthesis. In the present study, we provide the first evidence that the P2X7 receptor interacts with CD44 on the CHO-K1 cell surface. Thus, these data demonstrated that GAGs positively modulate the P2X7 receptor, and sCD44 is a part of a regulatory positive feedback loop linking P2X7 receptor activation for the intracellular response mediated through P2X7 receptor stimulation.

Highlights

In the absence of adenosine 5′-triphosphate (ATP) or 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP), even the highest heparin concentration did not elicit purinergic receptor P2X (P2X7) stimulation, and at saturation the agonist concentration (BzATP 4100 μM or ATP 44 mM) of heparin induced no further increase in Ca2+ influx. These results showed that heparin markedly sensitized Ca2+ entry through P2X7 at lower agonist concentrations (ATP and BzATP)
We present evidence for a positive allosteric modulation of P2X7 through GAGs in CHO cells. These data strongly suggest that GAGs from the cell surface bind to the P2X7 receptor and thereby facilitates the binding of ATP to the ligand-gated cation channel
The formation, recruitment and dilation of the P2X7 pore augmented in the presence of GAGs as demonstrated through the acceleration of cellular uptake of the large molecule propidium iodide (PI) (MW 668) and molecular dynamic simulations

Summary

Introduction

Classical pharmacology postulates that receptor activation occurs through drug-receptor interactions.[1,2] Advances achieved during the last years have increased the knowledge concerning this activation process and introduced new aspects that might modulate drug-receptor interactions, for example, receptor states, oligomerization with other proteins and allosteric mechanisms.[3]Extracellular matrix components, such as glycosaminoglycans (GAGs) and proteoglycans have shown the modulation of receptor activity.[4,5] The action of heparin on the muscarinic receptor M3 occurred through the heparin/extracellular matrix molecules/integrin complex, which positively modulates the M3 receptor, inducing relaxation through increased nitric oxide levels.[6]. Classical pharmacology postulates that receptor activation occurs through drug-receptor interactions.[1,2] Advances achieved during the last years have increased the knowledge concerning this activation process and introduced new aspects that might modulate drug-receptor interactions, for example, receptor states, oligomerization with other proteins and allosteric mechanisms.[3]. Extracellular matrix components, such as glycosaminoglycans (GAGs) and proteoglycans have shown the modulation of receptor activity.[4,5] The action of heparin on the muscarinic receptor M3 occurred through the heparin/extracellular matrix molecules/. The type III transforming growth factor-β (TGF-β) receptor interacts with heparan sulfate proteoglycan and other GAG chains.[11]

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