title: Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins.
abstract: Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.
authors.affiliation: Division of Digestive Diseases, Department of Medicine, Emory University, Atlanta, GA 30322, USA.
authors.forename: Lixin
authors.initials: L
authors.lastname: Wang
authors.forename: Vasantha
authors.initials: V
authors.lastname: Kolachala
authors.forename: Baljit
authors.initials: B
authors.lastname: Walia
authors.forename: Srividya
authors.initials: S
authors.lastname: Balasubramanian
authors.forename: Randy A
authors.initials: RA
authors.lastname: Hall
authors.forename: Didier
authors.initials: D
authors.lastname: Merlin
authors.forename: Shanthi V
authors.initials: SV
authors.lastname: Sitaraman
mesh_terms: D000241:Adenosine / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D020033:Protein Isoforms / Q000819:agonists / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D058906:Purinergic P1 Receptor Agonists; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D050600:SNARE Proteins; D015398:Signal Transduction / Q000502:physiology; D014018:Tissue Distribution; D033921:Vesicular Transport Proteins / Q000502:physiology*
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D020033:Protein Isoforms; D058906:Purinergic P1 Receptor Agonists; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D018047:Receptors, Purinergic P1; C100493:SNAP23 protein, human; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D000241:Adenosine