Rab27a negatively regulates CFTR chloride channel function in colonic epithelia: involvement of the effector proteins in the regulatory mechanism.
Cystic fibrosis, an autosomal recessive disorder, is caused by the disruption of biosynthesis or function of CFTR. CFTR regulatory mechanisms include channel transport to plasma membrane and protein-protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The colorectal epithelial HT-29 cells natively express CFTR and respond to cAMP with an increase in CFTR-mediated currents. DPC-inhibited currents could be completely eliminated with CFTR-specific SiRNA. Over-expression of Rab27a inhibited, while isoform specific SiRNA and Rab27a antibody stimulated CFTR-mediated currents in HT-29 cells. CFTR activity is inhibited both by Rab27a (Q78L) (constitutive active GTP-bound form of Rab27a) and Rab27a (T23N) (constitutive negative form that mimics the GDP-bound form). Rab27a mediated effects could be reversed by Rab27a-binding proteins, the synaptotagmin-like protein (SLP-5) and Munc13-4 accessory protein (a putative priming factor for exocytosis). The SLP reversal of Rab27a effect was restricted to C2A/C2B domains while the SHD motif imparted little more inhibition. The CFTR-mediated currents remain unaffected by Rab3 though SLP-5 appears to weakly bind it. The immunoprecipitation experiments suggest protein-protein interactions between Rab27a and CFTR. Rab27a appears to impair CFTR appearance at the cell surface by trapping CFTR in the intracellular compartments. Munc13-4 and SLP-5, on the other hand, limit Rab27a availability to CFTR, thus minimizing its effect on channel function. These observations decisively prove that Rab27a is involved in CFTR channel regulation through protein-protein interactions involving Munc13-4 and SLP-5 effector proteins, and thus could be a potential target for cystic fibrosis therapy.
Saxena
Sunil K
SK
Center for Cell and Molecular Biology, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030, USA. ssaxena@stevens.edu
Kaur
Simarna
S
D002352:Carrier Proteins / Q000502:physiology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D019073:HT29 Cells; D006801:Humans; D007413:Intestinal Mucosa / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology; D020691:rab GTP-Binding Proteins / Q000502:physiology*; D000075745:rab27 GTP-Binding Proteins
C505032:CFTR protein, human; D002352:Carrier Proteins; D008565:Membrane Proteins; C457772:SYTL5 protein, human; C507969:UNC13D protein, human; D000075745:rab27 GTP-Binding Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C066694:RAB27A protein, human; D020691:rab GTP-Binding Proteins