Regulation of CFTR trafficking by its R domain - PubMed (original) (raw)
Regulation of CFTR trafficking by its R domain
Christopher M Lewarchik et al. J Biol Chem. 2008.
Abstract
Phosphorylation of the R domain is required for cystic fibrosis transmembrane conductance regulator (CFTR) channel gating, and cAMP/protein kinase A (PKA) simulation can also elicit insertion of CFTR into the plasma membrane from intracellular compartments (Bertrand, C. A., and Frizzell, R. A. (2003) Am. J. Physiol. 285, C1-C18). We evaluated the structural basis of regulated CFTR trafficking by determining agonist-evoked increases in plasma membrane capacitance (Cm) of Xenopus oocytes expressing CFTR deletion mutants. Expression of CFTR as a split construct that omitted the R domain (Deltaamino acids 635-834) produced a channel with elevated basal current (Im) and no DeltaIm or trafficking response (DeltaCm) upon cAMP/PKA stimulation, indicating that the structure(s) required for regulated CFTR trafficking are contained within the R domain. Additional deletions showed that removal of amino acids 817-838, a 22-amino acid conserved helical region having a net charge of -9, termed NEG2 (Xie, J., Adams, L. M., Zhao, J., Gerken, T. A., Davis, P. B., and Ma, J. (2002) J. Biol. Chem. 277, 23019-23027), produced a channel with regulated gating that lacked the agonist-induced increase in CFTR trafficking. Injection of NEG2 peptides into oocytes expressing split DeltaNEG2 CFTR prior to stimulation restored the agonist-evoked DeltaCm, consistent with the concept that this sequence mediates the regulated trafficking event. In support of this idea, DeltaNEG2 CFTR escaped from the inhibition of wild type CFTR trafficking produced by overexpression of syntaxin 1A. These observations suggest that the NEG2 region at the C terminus of the R domain allows stabilization of CFTR in a regulated intracellular compartment from which it traffics to the plasma membrane in response to cAMP/PKA stimulation.
Figures
FIGURE 1.
Im (A) and Cm (B) recordings from a WT CFTR expressing oocyte during stimulation by bath addition of isoproterenol (10 μ
m
), see horizontal lines. Vm was held at -30 mV, and Im was obtained during a 200-mS pulse to -60 mV. Cm was calculated from currents recorded during a 10-mV hyperpolarizing voltage pulse, and panels a and b of A provide current recordings obtained from pulses before and after isoproterenol stimulation at the times indicated in the current record. The red lines show the fit of the current transient used to obtain Cm as described under “Experimental Procedures,” where Iss is the steady-state current, and Ipp is the peak point of the current transient. Each oocyte was injected with 1 ng of WT CFTR plus 1 ng of β2-adrenergic receptor cRNAs in 50 nl of water. C, average basal and stimulated Im and Cm values are from β2-adrenergic receptor (β_2-AR_)-injected oocytes (N = 3; n = 10) or oocytes expressing WT CFTR plus β2-adrenergic receptor (N = 4; n = 15). A value of p < 0.05 is considered statistically significant and is shown by an asterisk in all experiments.
FIGURE 2.
Current, capacitance, and cell surface expression of EXT-CFTR are increased by cAMP/PKA stimulation. A, Im and_Cm_ were obtained from Xenopus oocytes injected with 10 ng of EXT-CFTR and 1 ng of β2-adrenergic receptor cRNA 3 days prior to measurements (n = 4; n = 17). Mean luminometry values from oocytes (B) or HEK 293 cells (C) expressing EXT-CFTR (10 ng of cRNA or 4 μg of cDNA/35-mm plate, respectively) are shown. Values were background-subtracted and normalized to mean values obtained under basal, nonstimulated conditions (oocytes,N = 4 and n = 20; HEK cells, N = 5 and n = 20).
FIGURE 3.
The functional half-life of CFTR is reduced during cAMP/PKA stimulation. Current decay curves were produced by continuous incubation of oocytes expressing WT-CFTR (1 ng) and β2-adrenergic receptor (1 ng) with brefeldin A (10 μ
m
). The data were fit with a single exponential function using SigmaPlot (Systat Software). A, oocytes were stimulated continuously with 10 μ
m
isoproterenol, and currents were recorded at 0, 2, 4, 8, and 24 h (N = 5; n = 20, each data point). B, currents were recorded from oocytes at separate time points (0, 1, 2, 4, 8, and 24 h) after initial treatment with 10 μ
m
BFA. Current measurements were obtained at maximal stimulation following isoproterenol stimulation at the indicated times by_arrowheads_ (N = 4; n = 16).
FIGURE 4.
The functional properties of split CFTR constructs resemble those of WT CFTR. Oocytes were co-injected with 0.5 ng of the CFTR half-channel cRNAs (1–834 + 835–1480 (N = 4; n = 16) or 1–634 + 635–1480 (N = 3; n = 10) and 1 ng of β2-adrenergic receptor, and Im and_Cm_ were recorded in response to 10 μ
m
isoproterenol, as in Fig. 1.
FIGURE 5.
CFTR lacking the R domain does not exhibit regulated current or capacitance responses. A and B, time courses of_Im_ and Cm in an oocyte injected with ΔR-N/C cRNAs (0.5 ng each, 1–634 + 835–1480) plus 1 ng of β2-adrenergic receptor. Horizontal line indicates addition of 10 μ
m
isoproterenol. C, summary_Im_ and Cm data for ΔR-N/C expressing oocytes (N = 6; n = 29).
FIGURE 6.
Expression of the R domain does not induce regulated behavior of ΔR-N/C. cRNA expression conditions are as follows: 0.5 ng each of ΔR-N/C cRNAs, with or without 5 ng of R domain, plus 1 ng of β2-adrenergic receptor cRNA. A, mean basal and stimulated Im and Cm values ΔR-N/C (N = 3; n = 14) and ΔR-N/C + R domain (N = 3; n = 17). The source of the difference in mean_Cm_ values of these groups is not known; however, these values are within the range observed, and variation does arise from oocyte size and membrane folding (32). B, immunoblot of lysates from oocytes expressing ΔR-N/C or ΔR-N/C + R domain; expression conditions as in A. Membranes were probed with R domain-specific anti-CFTR monoclonal antibody (1:1000); see “Experimental Procedures” for protocol.
FIGURE 7.
Partial R domain deletions mimic the absence of a trafficking response in ΔR-N/C. A and B, Im and_Cm_ recordings of split CFTR (1–784 + 835–1480) co-expressed with β2-adrenergic receptor; all cRNAs are 1 ng/oocyte. Horizontal line indicates the addition of 10 μ
m
isoproterenol. C, summary data for 1–784 + 835–1480 CFTR (N = 3; n = 11). D, summary data for split ΔNEG2 injected oocytes; cRNAs 1 ng/oocyte (N = 4; n = 16).
FIGURE 8.
Peptide injection restores regulated trafficking to ΔNEG2. A, summary data for ΔNEG2 expressing oocytes injected with 23 nl of 50 m
m
NEG2 peptide (∼1 nmol) prior to_Im_ and Cm recordings (N = 4;n = 14); cRNA is 7 ng/oocyte. B, summary data for changes in_Cm_ (%) evoked by 10 μ
m
isoproterenol following injection of the indicated peptide into oocytes expressing split ΔNEG2 CFTR (as in A). Sequences of peptides: NEG2, GLEISEEINEEDLKECFFDDME; sNEG2, LIKEFSEEDGECLMIDEDENEF; hNEG2, GLEISEQINQQNLKQSFFNDME. See Ref. for discussion of peptide structures as determined by circular dichroism. sNEG2, N = 3 and_n_ = 12; hNEG2, N = 4 and n = 16.
FIGURE 9.
hNEG2-CFTR retains regulated trafficking properties. A, Im and Cm summary data for hNEG2-CFTR (N = 3; n = 13). B, Western blot of HEK 293 cells transfected with WT CFTR, hNEG2-CFTR, or sNEG2-CFTR. Cells were transfected with 4 μg of cDNA for each construct; after 24 h, cells were harvested and subjected to immunoblot using monoclonal antibody directed against the CFTR C terminus (1:2500). Results are typical of three experiments.
FIGURE 10.
Deletion of NEG2 region eliminates the effect of syntaxin 1A on CFTR-mediated ΔIm and ΔCm. cRNA injections: WT-CFTR (1 ng), ΔR-N/C (1 ng total), or ΔNEG2 (7.5 ng) with or without S1A (10 ng), as indicated, plus β2-adrenergic receptor (1 ng); stimulation with 10 μ
m
isoproterenol.*, significant difference between basal and stimulated conditions (p < 0.05); ++ indicates a significant difference with S1A co-expression (p < 0.05). N = 2; n = 9, all groups.
References
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