Expression levels strongly affect ligand-induced sequestration of B2 bradykinin receptors in transfected cells.
Transfection of cells with expression vectors is one of the most important tools used to assess the effects of receptor mutations on ligand-induced receptor sequestration. Most transfection methods give rise to transiently or stably transfected clones with a wide range of receptor expression levels that may also depend on the mutations made. It is, therefore, important to determine how the regulation of the receptors depends on their numbers per cell. In Chinese hamster ovary (CHO) and human embryonic kidney (HEK)-293 cells expressing high levels of B(2) kinin receptors, we observed poor sequestration indicated by <20% reduction in cell surface receptor number after 10 min of stimulation with 1 microM bradykinin (BK) compared with >70% in low-expressing cells. Whereas the rate of [(3)H]BK internalization (internalized [(3)H]BK in percentage of total bound [(3)H]BK) in low-expressing cells was independent of the ligand-concentration used, in high-expressing cells a strong rate decrease was observed with higher (>1 nM) concentrations. Lower ligand concentrations, however, led to internalization rates identical to those obtained in low-expressing cells. Transiently transfected HEK and COS-7 cells showed results similar to those of stably high-expressing cells. Our results demonstrate the difficulty in determining the internalization pattern of (mutated) B(2) kinin receptors, and possibly of G protein-coupled receptors in general, using a sequestration assay in high-expressing cells or transiently transfected cells with high numbers of receptors per transfected cell. However, the receptor (mutant)-specific internalization rate can be measured, provided that the ligand concentrations used are below a threshold at which the internalization rate is still independent of the ligand concentration.
Ludwig-Maximilians-Universitaet Muenchen, Abteilung Klinische Chemie und Klinische Biochemie, Germany. Alexander.Faussner@clinbio.med.uni-muenchen.de
Alexander
A
Faussner
Alexandra
A
Bauer
Irina
I
Kalatskaya
Marianne
M
Jochum
Hans
H
Fritz
D000818:Animals; D001920:Bradykinin / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006224:Cricetinae; D018274:Electroporation; D005822:Genetic Vectors; D006801:Humans; D008024:Ligands; D009154:Mutation / Q000235:genetics; D011506:Proteins / Q000378:metabolism; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000096:biosynthesis* / Q000235:genetics*; D014162:Transfection
D008024:Ligands; D011506:Proteins; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D001920:Bradykinin