Localisation of endothelin B receptor variants to plasma membrane microdomains and its effects on downstream signalling.
The endothelin B (ET(B)) receptor can undergo a proteolytic cleavage resulting in an unglycosylated N-terminally truncated receptor. We investigated whether ET(B) receptor processing affects caveolar localisation and mitogenic signalling. Distinct subcellular localisations of ET(B) receptor constructs and epidermal growth factor (EGF) receptor ligands were analysed performing detergent-free caveolae preparations and total internal reflection fluorescence microscopy. ET(B) receptor-induced transactivation of the EGF receptor and its downstream signalling was investigated performing shedding assays and ERK1/2 phosphorylation analyses. In COS7 cells, the N-terminally truncated but not the full-length or glycosylation-deficient ET(B) receptor localised to caveolae. In caveolae-free HEK293 cells, only ET(B) receptor constructs fused to caveolin-2 localised to membrane microdomains. A caveolar accumulation of the ET(B) receptor disfavoured EGF receptor ligand shedding. Nonetheless, the activation of ERK1/2 was efficient and long-lasting. In HEK293 cells, the shedding activity was also impaired by N-terminal truncation. The subsequent ERK1/2 phosphorylation was long-lasting only for the full-length ET(B) receptor. We conclude that the ET(B) receptor localisation might depend on the presence of caveolae within the cell investigated. The data further suggest that caveolar enrichment of ET(B) receptors does not facilitate the release of EGF receptor ligands. However, independent of their localisation, ET(B) receptors are able to induce an ERK1/2 phosphorylation.
Grossmann
Solveig
S
Rudolf-Boehm-Institut für Pharmakologie und Toxikologie, Medizinische Fakultät, Unversität Leipzig, Härtelstrasse 16-18, Leipzig, Germany.
Higashiyama
Shigeki
S
Oksche
Alexander
A
Schaefer
Michael
M
Tannert
Astrid
A
D000818:Animals; D019556:COS Cells; D021941:Caveolae / Q000378:metabolism; D051242:Caveolin 1 / Q000378:metabolism; D051243:Caveolin 2 / Q000378:metabolism; D002460:Cell Line; D002522:Chlorocebus aethiops; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D044023:Receptor, Endothelin B / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D051242:Caveolin 1; D051243:Caveolin 2; D044023:Receptor, Endothelin B; D066246:ErbB Receptors; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3