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Internalization and down-regulation of the prostacyclin receptor in human platelets.

ABSTRACT: The internalization of [3H]iloprost, a prostacyclin analogue, was studied in human platelets by binding studies. After incubation with [3H]iloprost at 37 degrees C, addition of unlabelled ligand at either 37 degrees C or 4 degrees C caused dissociation of 74% and 52% of the bound ligand respectively, suggesting that a portion had been internalized. The percentage of [3H]iloprost bound at equilibrium to the surface (evaluated by acid treatment) at either 37 degrees C or 4 degrees C was markedly different (80% versus 25%). Internalization was dependent on time and on the ligand nature and concentration. Energy-depleting agents (dinitrophenol and 2-deoxyglucose) completely inhibited internalization, whereas probenecid (inhibitor of organic anion transporters) did not affect it significantly. Subcellular fractionation indicated that, at 4 degrees C or in the absence of ligand, most of the receptor was present in membrane fractions (pellet at 27000 or 105000 g), whereas, when platelets were preincubated at 37 degrees C with iloprost, the receptor was found mainly in the cytosolic fraction. In platelets preincubated with iloprost at 4 degrees C, two classes of binding sites were present, whereas after preincubation at 37 degrees C only the lower-affinity sites were detected. After exposure to the agonist, iloprost-induced inhibition of platelet aggregation and activation of adenylate cyclase and cAMP production were significantly lower. Taken together, these data demonstrate that human platelets can internalize a high-affinity binding site for iloprost, presumably the prostacyclin receptor.

SUBMITTER: Giovanazzi S

PROVIDER: S-EPMC1218530 | biostudies-other | 1997 Jul

REPOSITORIES: biostudies-other

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Project description:The ?2-adrenergic receptor (?2AR) plays important physiological roles in the heart and lung and is the primary target of ?-agonists, the mainstay asthma drugs. Activation of ?2AR by ?-agonists is attenuated by receptor down-regulation, which ensures transient stimulation of the receptor but reduces the efficacy of ?-agonists. Here we report the identification, through a functional genome-wide RNA interference (RNAi) screen, of new genes critically involved in ?2AR down-regulation. We developed a lentivirus-based RNAi library consisting of 26-nt short-hairpin RNAs (shRNAs). The library was generated enzymatically from a large collection of expressed sequence tag (EST) DNAs corresponding to ?20,000 human genes and contains on average ?6 highly potent shRNAs (>75% knockdown efficiency) for each gene. Using this novel shRNA library, together with a robust cell model for ?2AR expression, we performed fluorescence-activated cell sorting and isolated cells that, as a consequence of shRNA-mediated gene inactivation, exhibited defective agonist-induced down-regulation. The screen discovered several previously unrecognized ?2AR regulators, including farnesyl diphosphate synthase (FDPS). We showed that inactivation of FDPS by shRNA, small interfering RNA, or the highly specific pharmaceutical inhibitor alendronate inhibited ?2AR down-regulation. Notably, in human airway smooth muscle cells, the physiological target of ?-agonists, alendronate treatment functionally reversed agonist-induced endogenous ?2AR loss as indicated by an increase in cAMP production. FDPS inactivation interfered with ?2AR internalization into endosomes through disrupting the membrane localization of the Rab5 small GTPase. Furthermore, Rab5 overexpression reversed the deficient receptor down-regulation induced by alendronate, suggesting that FDPS regulates receptor down-regulation in a Rab5-dependent manner. Together, our findings reveal a FDPS-dependent mechanism in the internalization and down-regulation of ?2AR, identify FDPS as a potential target for improving the therapeutic efficacy of ?-agonists, and demonstrate the utility of the unique EST-derived shRNA library for functional genetics studies.

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Internalization and down-regulation of the prostacyclin receptor in human platelets.

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