Mutation of a Dibasic Amino Acid Motif Within the C Terminus of the P2X7 Nucleotide Receptor Results in Trafficking Defects and Impaired Function

Denlinger, Loren C.; Sommer, Julie A.; Parker, K.; Gudipaty, Lalitha; Fisette, Philip L.; Watters, Jyoti W.; Proctor, Richard A.; Dubyak, George R.; Bertics, Paul J.

doi:10.4049/jimmunol.171.3.1304

Cited by 71 publications

(70 citation statements)

References 43 publications

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“…The conserved YXXXK motif found in Cterminal of P2XRs other than P2X7R is necessary for their expression at the cell surface [24]. In human P2X7R, a motif located in the distal region of its C-terminus tail is necessary for proper receptor trafficking; progressive deletions of this tail between residues 551 and 581 are nonfunctional and are not found in the plasma membrane [10,41]. The same motif is also involved in pore dilation [45,48].…”

Section: Discussionmentioning

confidence: 99%

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

Jindrichova

Kuzyk

Li³

et al. 2012

Purinergic Signalling

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The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single-and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-μM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.

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Section: Discussionmentioning

confidence: 99%

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

Jindrichova

Kuzyk

Li³

et al. 2012

Purinergic Signalling

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“…Mutations in the region also alter the permeability of P2X receptor channels for divalent ions [86]. The second membrane-spanning domain is also involved in protein folding and assembly of P2X subunits [87,88].…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

Sun

Chu

Singh

et al. 2009

Purinergic Signalling

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The P2X 7 receptor exhibits significant allelic polymorphism in humans, with both loss and gain of function variants potentially impacting on a variety of infectious and inflammatory disorders. At least five loss-offunction polymorphisms (G150R, R307Q, T357S, E496A, and I568N) and two gain-of-function polymorphisms (H155Y and Q460R) have been identified and characterized to date. In this study, we used RT-PCR cloning to isolate and characterize P2X 7 cDNA clones from human PBMCs and THP-1 cells. A previously unreported variant with substitutions of V80M and A166G was identified. When expressed in HEK293 cells, this variant exhibited heightened sensitivity to the P2X 7 agonist (BzATP) relative to the most frequent allele, as shown by pore formation measured by fluorescent dye uptake into cells. Mutational analyses showed that A166G alteration was critical for the gain-offunction change, while V80M was not. Full-length variants with multiple previously identified nonsynonymous SNPs (H155Y, H270R, A348T, and E496A) were also identified. Distinct functional phenotypes of the P2X 7 variants or mutants constructed with multiple polymorphisms were observed. Gain-of-function variations (A166G or H155Y) could not rescue the loss-of-function E496A polymorphism. Synergistic effects of the gain-of-function variations were also observed. We also identified the A348T alteration as a weak gain-of-function variant. Thus, these results identify the new gain-of-function variant A166G and demonstrate that multiple-gene polymorphisms contribute to functional phenotypes of the human P2X 7 receptor.Furthermore, the results demonstrate that the C-terminal of the cysteine-rich domain 1 of P2X 7 is critical for regulation of P2X 7 -mediated pore formation.

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“…However, P2X 7 Rs have a long intracellular C-terminal end, being 120 -200 amino acids longer compared with other P2X receptors (3,17). This long C-terminal end has been proposed to be responsible for most of the specific biophysical properties of the P2X 7 R with numerous functions such as connecting the receptor to structures responsible for the large pore formation (3,17), recently proposed as being pannexin-1 (18), the interaction with adaptor and signaling proteins (19,20), the regulation of P2X 7 R trafficking to the plasma membrane (21,22), and the modulation of channel gating (23). Recently, we demonstrated that rat P2X 7 R C terminus is also displaying a novel 1-5-16 calmodulin-binding motif responsible for a calcium-dependent facilitation (24).…”

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confidence: 99%

C-terminal Calmodulin-binding Motif Differentially Controls Human and Rat P2X7 Receptor Current Facilitation

Roger

Gillet

Baroja‐Mazo

et al. 2010

Journal of Biological Chemistry

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P2X 7 receptors (P2X 7 R) are ATP-gated calcium-permeable cationic channels structurally unique among the P2X family by their much longer intracellular C-terminal tail. P2X 7 Rs show several unusual biophysical properties, in particular marked facilitation of currents and leftward shift in agonist affinity in response to repeated or prolonged agonist applications. We previously found the facilitation at rat P2X 7 R resulted from a Ca 2؉ -calmodulin-dependent process and a distinct calcium-independent process. However, P2X 7 Rs show striking species differences; thus, this study compared the properties of ATPevoked facilitation of currents in HEK293 cells transiently expressing the human or rat P2X 7 R as well as rat/human, human/rat chimeric, and mutated P2X 7 Rs. Facilitation at the human P2X 7 R was 5-fold slower than at the rat P2X 7 R. Facilitation did not resulting from an increase of receptor addressing the plasma membrane. We found the human P2X 7 R shows only calcium-independent facilitation with no evidence for calmodulin-dependent processes, nor does it contain the novel 1-5-16 calmodulin binding domain present in the C terminus of rat P2X 7 R. Replacement of three critical residues of this binding domain from the rat into the human P2X 7 R (T541I, C552S, and G559V) reconstituted the Ca 2؉ -calmodulin-dependent facilitation, leaving the calcium-independent facilitation unaltered. The leftward shift in the ATP concentration-response curve with repeated agonist applications appears to be a property of the calcium-independent facilitation process because it was not altered in any of the chimeric or mutated P2X 7 Rs. The absence of Ca 2؉ -dependent facilitation at the human P2X 7 R may represent a protective adaptation of the innate immune response in which P2X 7 R plays significant roles.

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Mutation of a Dibasic Amino Acid Motif Within the C Terminus of the P2X7 Nucleotide Receptor Results in Trafficking Defects and Impaired Function

Cited by 71 publications

References 43 publications

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

C-terminal Calmodulin-binding Motif Differentially Controls Human and Rat P2X7 Receptor Current Facilitation

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