title: Cell-cell fusion and internalization of the CNS-based, HIV-1 co-receptor, APJ.
abstract: APJ, a member of the human G protein-coupled seven-transmembrane receptor family, has been shown to serve as a coreceptor for the entry of human immunodeficiency virus type I (HIV-1) and simian immunodeficiency virus (SIV), and it is dramatically expressed in central nervous system (CNS)-based cells. In this study, expression of APJ tagged with the green fluorescent protein (GFP) and a fluorescent peptide, 5-carboxyfluorescein (5-CF) conjugated Apelin-13, were utilized for studying receptor internalization and recycling, in stably expressing indicator cells, human neurons, primary CNS microvascular endothelial cells (MVECs), and astrocytes. Fusion of the C-terminus of APJ to the N-terminus of GFP did not alter receptor ligand binding and functions, including signaling and internalization. Using 293 cells stably expressing APJ-GFP, we demonstrated that rapid internalization of the APJ receptor was induced by stimulation with Apelin-36 and Apelin-13, in a dose-dependent manner. Furthermore, investigations showed that the internalized APJ was colocalized with transferrin receptors, suggesting that the internalization of APJ induced by Apelin is likely to be via clathrin-coated pits. Interestingly, we found that the internalized APJ molecules were recycled to the cell surface within 60 min after removal of Apelin-13, but most of the internalized APJ still remained in the cytoplasm, even 2 h after washout of Apelin-36. The intact cytoplasmic C-terminal domain was found to be required for ligand-induced APJ internalization. Human neurons were dramatically stained by the APJ-binding fluorescent peptides. Primary human fetal astrocytes were less strongly labeled with 5-CF-Apelin-13, and in primary human CNS MVECs only weak distribution of green fluorescence specific for APJ in the cytoplasm was observed. Apelin-36 blocked cell membrane fusion mostly due to steric interference, with only a very modest effect on receptor internalization. The CNS represents a unique reservoir site for HIV-1. As such, molecular therapeutics and small molecular inhibitors of HIV-1 entry via this unique CNS receptor are now able to be rationally designed.
authors.affiliation: The Dorrance H. Hamilton Laboratories, Thomas Jefferson University, Jefferson Medical College, Center for Human Virology, Division of Infectious Diseases, Department of Medicine, 1020 Locust Street, Suite 329, Philadelphia, PA 19107, USA.
authors.forename: Naiming
authors.initials: N
authors.lastname: Zhou
authors.forename: Xuejun
authors.initials: X
authors.lastname: Fan
authors.forename: Muhammad
authors.initials: M
authors.lastname: Mukhtar
authors.forename: Jianhua
authors.initials: J
authors.lastname: Fang
authors.forename: Charvi A
authors.initials: CA
authors.lastname: Patel
authors.forename: Garrett C
authors.initials: GC
authors.lastname: DuBois
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Pomerantz
mesh_terms: D000818:Animals; D000075244:Apelin Receptors; D001253:Astrocytes / Q000821:virology; D001921:Brain / Q000821:virology*; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002459:Cell Fusion; D002478:Cells, Cultured; D006224:Cricetinae; D004730:Endothelium, Vascular / Q000821:virology; D005333:Fetus; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008833:Microcirculation; D009474:Neurons / Q000821:virology; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled; D011991:Receptors, Virus / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018243:Teratocarcinoma; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: C104575:APLNR protein, human; D000075244:Apelin Receptors; D008164:Luminescent Proteins; D017448:Receptors, Dopamine D2; D043562:Receptors, G-Protein-Coupled; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002118:Calcium