Cleavage of the angiotensin II type 1 receptor and nuclear accumulation of the cytoplasmic carboxy-terminal fragment.
Our published studies show that the distribution of the ANG II type 1 (AT(1)) receptor (AT(1)R), expressed as a enhanced yellow fluorescent fusion (YFP) protein (AT(1)R/EYFP), is altered upon cellular treatment with ANG II or coexpression with intracellular ANG II. AT(1)R accumulates in nuclei of cells only in the presence of ANG II. Several transmembrane receptors are known to accumulate in nuclei, some as holoreceptors and others as cleaved receptor products. The present study was designed to determine whether the AT(1)R is cleaved before nuclear transport. A plasmid encoding a rat AT(1)R labeled at the amino terminus with enhanced cyan fluorescent protein (CFP) and at the carboxy terminus with EYFP was employed. Image analyses of this protein in COS-7 cells, CCF-STTG1 glial cells, and A10 vascular smooth muscle cells show the two fluorescent moieties to be largely spatially colocalized in untreated cells. ANG II treatment, however, leads to a separation of the fluorescent moieties with yellow fluorescence accumulating in more than 30% of cellular nuclei. Immunoblot analyses of extracts and conditioned media from transfected cells indicate that the CFP domain fused to the extracellular amino-terminal AT(1)R domain is cleaved from the membrane and that the YFP domain, together with the intracellular cytoplasmic carboxy terminus of the AT(1)R, is also cleaved from the membrane-bound receptor. The carboxy terminus of the AT(1)R is essential for cleavage; cleavage does not occur in protein deleted with respect to this region. Overexpressed native AT(1)R (nonfusion) is also cleaved; the intracellular 6-kDa cytoplasmic domain product accumulates to a significantly higher level with ANG II treatment.
Cook
Julia L
JL
Ochsner Clinic Foundation, Ochsner Health System, 1516 Jefferson Hwy., New Orleans, LA 70121, USA. jcook@ochsner.org
Mills
Sarah J
SJ
Naquin
Ryan T
RT
Alam
Jawed
J
Re
Richard N
RN
D000804:Angiotensin II / Q000502:physiology*; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D045726:Metalloproteases / Q000037:antagonists & inhibitors; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000166:cytology; D009457:Neuroglia / Q000166:cytology; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D008164:Luminescent Proteins; D044140:Receptor, Angiotensin, Type 1; D011993:Recombinant Fusion Proteins; D000804:Angiotensin II; D045726:Metalloproteases