Calreticulin in rainbow trout: a limited response to endoplasmic reticulum (ER) stress.
Calreticulin (CRT) is a resident protein of the endoplasmic reticulum where it serves as a calcium modulator and chaperone to newly synthesized glycoproteins. In mammals, CRT is a structurally conserved 46 kDa protein that demonstrates anomalous migration at 60 kDa on SDS polyacrylamide gels and can be up-regulated by A23187 and thapsigargin due to the endoplasmic reticulum stress elements (ERSE) in the promoter region of its gene. CRT has numerous proposed functions and has been localized to the surface of PHA-stimulated T lymphocytes. CRT has been identified in mammals, plants and more recently from rainbow trout. Here, we report the cloning of the CRT proximal promoter from rainbow trout which includes elements typical of genes transcribed by RNA polymerase II including a TATA box, an Sp1 binding site, CCAAT boxes and the conservation of promoter stress elements (ERSE) demonstrated to be responsible for calcium modulation in mammals. This report demonstrates that the anomalous 60 kDa gel migration of mammalian CRT is conserved in rainbow trout and that CRT exists primarily as a dimer or oligomer in all tissues tested, excluding muscle and sperm in which it exists as a single polypeptide. Although it contains a potential N-glycosylation site, rainbow trout CRT is not subject to N-type glycosylation. Through the use of reverse transcriptase (RT) PCR along with western blotting, in both primary cultured leukocytes and the macrophage cell line RTS11, this report demonstrates that, unlike mammals, rainbow trout CRT is not strongly up-regulated by the calcium homeostasis antagonists, A23187 and thapsigargin, but is present on the cell surface of PHA-stimulated leukocytes. Taken together, this data suggests that CRT may have an alternative mode of regulation or function in fish.
Kales
Stephen C
SC
Department of Biology, University of Waterloo, 200 University Ave. W, Waterloo, Ontario, Canada N2L 3G1.
Bols
Niels C
NC
Dixon
Brian
B
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D000954:Antigens, Surface / Q000378:metabolism; D001483:Base Sequence; D002121:Calcium Channel Blockers / Q000494:pharmacology; D037282:Calreticulin / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015870:Gene Expression; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D020218:Response Elements; D013312:Stress, Physiological / Q000235:genetics / Q000378:metabolism*
D000906:Antibodies; D000954:Antigens, Surface; D002121:Calcium Channel Blockers; D037282:Calreticulin