C-terminal fusion of eGFP to the bradykinin B2 receptor strongly affects down-regulation but not receptor internalization or signaling.
A functional comparison was made between the wild-type bradykinin B2 receptor (B2wt) and the chimera B2eGFP (enhanced green-fluorescent protein fused to the C-terminus of B2wt), both stably expressed in HEK 293 cells. There was almost no difference in terms of ligand-inducible receptor phosphorylation and internalization, signal transduction (accumulation of inositol phosphates) or expression and affinity. However, stimulation for up to 8 h with 10 microM bradykinin (BK) resulted in a strong decrease in surface receptors (by 60% within 5 h) in B2wt, but not in B2eGFP. When the expression levels of both constructs where comparably reduced using a weaker promoter, long-term stimulation resulted in a reduction in surface receptors for B2wt(low) to less than 20% within 1 h, whereas the chimera B2eGFP(low) still displayed 50% binding activity after 2 h. A 1-h incubation in the absence of BK resulted in a recovery of 60% of the binding in B2wt(low) after 1-h stimulation with BK, but of only 20% after 7-h stimulation. In contrast, B2eGFP(low) levels were restored to more than 70%, even after 7-h stimulation. These data indicate that although the fusion of eGFP to B2wt does not affect its ligand-induced internalization, it strongly reduces the down-regulation, most likely by promoting receptor recycling over degradation.
Kalatskaya
Irina
I
Abteilung für Klinische Chemie und Klinische Biochemie, Ludwig-Maximilians-Universität München, Nussbaumstrasse 20, D-80336 München, Germany.
Schüssler
Steffen
S
Seidl
Cornelia
C
Jochum
Marianne
M
Faussner
Alexander
A
D001920:Bradykinin / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D021381:Protein Transport / Q000187:drug effects; D043782:Receptor, Bradykinin B2 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013997:Time Factors; D014162:Transfection / Q000379:methods
D007295:Inositol Phosphates; D010446:Peptide Fragments; D043782:Receptor, Bradykinin B2; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D001920:Bradykinin