Calreticulin is at the surface of circulating neutrophils and uses CD59 as an adaptor molecule.
Calreticulin, which has been proposed to be a C1q receptor on neutrophils, has neither a transmembrane domain nor a GPI-anchor attachment site and must utilize an adaptor molecule to attach to the plasma membrane. The expression of ecto-calreticulin on purified human neutrophils did not result from contamination by soluble or intracellular calreticulin released during cell fractionation because it was expressed on circulating neutrophils, and the expression did not increase significantly with neutrophil isolation. All neutrophils expressed calreticulin with a unimodal distribution. Treatment of neutrophils with either a cholesterol-binding agent or phosphatidylinositol-specific phospholipase C dramatically decreased ecto-calreticulin expression indicating that the adaptor molecule(s) are located in lipid rafts and have a GPI-anchor. Analysis for the co-expression of specific GPI-anchored proteins and ecto-calreticulin in cells that were deficient in specific GPI-anchored proteins, indicated that ecto-calreticulin was best associated with CD59. Calreticulin reciprocally immunoprecipited with CD59, which provided direct evidence that CD59 is an adaptor for ecto-calreticulin. Immunofluorescence and confocal microscopy demonstrated that ecto-calreticulin co-localized with a fraction of CD59 at the cell surface. Cross-linking ecto-calreticulin with antibodies induced a Ca2+ flux, which suggests that ecto-calreticulin is capable of signaling following ligand binding. Ecto-calreticulin has been associated with diverse cellular functions. An appreciation that the adaptors for ecto-calreticulin are GPI-anchored will provide a framework for understanding any common features underlying ecto-calreticulin ligation.
Ghiran
Ionita
I
Divisions of Allergy-Inflammation and Infectious Disease, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA. ighiran@bidmc.harvard.edu
Klickstein
Lloyd B
LB
Nicholson-Weller
Anne
A
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism*; D000906:Antibodies / Q000494:pharmacology; D018957:CD59 Antigens / Q000276:immunology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D037282:Calreticulin / Q000276:immunology / Q000378:metabolism*; D002458:Cell Fractionation; D003432:Cross-Linking Reagents / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006457:Hemoglobinuria, Paroxysmal / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D011233:Precipitin Tests; D017452:Receptors, IgG / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D010738:Type C Phospholipases / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D000906:Antibodies; D018957:CD59 Antigens; D037282:Calreticulin; D003432:Cross-Linking Reagents; D017261:Glycosylphosphatidylinositols; D017452:Receptors, IgG; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium