Abstract
Prostacyclin, the major cyclooxygenase-derived product of arachidonic acid formed in the vasculature, mediates its potent anti-thrombotic and anti-proliferative effects through its G protein-coupled receptor (GPCR) termed the IP. Unlike many GPCRs, agonist-induced internalization of the IP occurs in an arrestin/GPCR kinase-independent manner. However, deletion of the IP COOH-terminal region prevented internalization suggesting that protein interactions at this region are involved in IP regulation. Using the COOH-terminal region of IP as bait we identified the delta subunit of cGMP phosphodiesterase 6 (PDE6delta) as a novel hIP-interacting protein in two independent yeast two-hybrid screens. Interaction of IP and PDE6delta was confirmed by co-immunoprecipitation in HEK293 cells, and in HEPG2 cells, which endogenously express neither IP nor PDE6delta. IP isoprenylation was critical for this interaction, as PDE6delta was unable to associate with an isoprenylation-deficient mutant IP (IPSSLC). PDE6delta overexpression altered the temporal pattern of agonist-induced internalization of IP, but not IPSSLC, in HEPG2 cells, increasing initial internalization but facilitating the return of IP to the cell surface despite the continued presence of agonist. Depletion of PDE6delta using short interfering RNA abolished cicaprost-induced IP internalization in human aortic smooth muscle cells. Recycling of IP, but not IPSSLC, upon agonist removal was facilitated by overexpression of PDE6delta. Thus PDE6delta interacts specifically with IP to modulate receptor trafficking.
Highlights
His reporter expression was inhibited with 3-aminotriazol (3-AT), to titrate out the number of false positives
Desensitization is mediated through PKC-dependent phosphorylation (6), while IP sequestration occurred, in part, through a dynamin-dependent clathrin-coated vesicular pathway that did not employ arrestin as an adapter molecule (5)
Determinants for both regulatory steps are located in the COOH-terminal tail region of the IP, only in the case of PKC-dependent phosphorylation has the critical residue, Ser[328], been identified (6). This particular site was not required for agonist-dependent sequestration of the IP, the COOH-terminal tail of the receptor proved indispensable for this regulatory step to proceed (5), suggesting that this region may provide a docking site for proteins involved in IP regulation (5)
Summary
His reporter expression was inhibited with 3-aminotriazol (3-AT), to titrate out the number of false positives. LacZ expression in surviving colonies was inferred from -galactosidase activity, assayed, as described (20), in a colony filter lift assay using 5-bromo-4-chloro-3-indolyl -D-galactoside as substrate. The cDNAs from LacZ-positive clones were sequenced across the Gal4/library cDNA boundary and analyzed using the BLAST algorithm at the NCBI (www.ncbi.nlm.nih.gov/BLAST/). Construct Generation and Site-directed Mutagenesis—Primers were designed to mutate Cys[383] to Ser[383] (sense oligonucleotide: 5Ј GCC AGC GTC GCC AGC TCC CTC TGC TGA TGG ATC C), in the hIP, triple tagged (3x) at its amino terminus with the hemagglutinin (HA) epitope tag (obtained from the University of Missouri-Rolla cDNA Resource Center). The mutated receptor was termed IPSSLC reflecting the change in the amino acid sequence of the last four amino acids of hIP from CSLC to SSLC.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
