Specific interaction between the hop1 intracellular loop 3 domain of the human PAC(1) receptor and ARF

The PAC(1), VPAC(1) and VPAC(2) receptors are members of the secretin (Group II) family of G protein-coupled receptors. All members of this family activate adenylate cyclase and several have also been shown to activate phospholipase C. We have recently reported that the rat VPAC(1), VPAC(2) and PAC(1) receptors activate phospholipase D and that distinct pathways are utilised by two intracellular loop 3 splice variants of PAC(1), one of which is ARF-dependent. Phospholipase D activation by the hop1, but not the null (short), form of the PAC(1) receptor is sensitive to brefeldin A, an inhibitor of GTP exchange at ARF. We have expressed the null and hop1 intracellular loop 3 domains of the human PAC(1) receptor in bacteria as GST-fusion proteins and used them as peptide affinity matrices to determine whether a functional interaction exists between these domains and ARF. Using this GST pull-down assay, we have shown binding of the small G protein ARF6 to the hop1 but not the null domain of this receptor.