Agonist-dependent trafficking of alpha2-adrenoceptor subtypes: dependence on receptor subtype and employed agonist.
Many G protein-coupled receptors (GPCRs) are internalized from the plasma membrane after agonist exposure. Previously, marked agonist-induced internalization of human alpha2A- and alpha2B-adrenergic receptors (AR) was observed in transfected neuronal rat pheochromocytoma (PC12) cells; alpha2A- and alpha2B-AR were internalized into partly distinct intracellular vesicles (Olli-Lähdesmäki et al., J. Neurosci. 19, 9281-9288, 1999). In this paper, the extent of alpha2-AR internalization was quantitated in human embryonic kidney (HEK-293) and PC12 cells by combined application of cell surface biotinylation and ELISA methods, which allow measurement of protein trafficking in intact, differentiated and undifferentiated cells. Significant subtype-specific (but not cell type-dependent) trafficking of human alpha2-AR was observed by quantitation and immunocytochemistry. Agonist-induced sequestration of alpha2B-AR was markedly reduced after blocking the formation of clathrin-coated vesicles by hyperosmotic sucrose pretreatment. The sequestration of alpha2A-AR was partly inhibited after sucrose pretreatment but could be further reduced after inhibiting the formation of both clathrin-coated and caveolin vesicles by combined pretreatment with hyperosmotic sucrose and filipin. Differences were also observed in the recycling of alpha2A- and alpha2B-AR. The extent of maximal agonist-induced sequestration in PC12 cells was not directly dependent on relative agonist efficacy.
Olli-Lähdesmäki
Tuire
T
Department of Pharmacology and Clinical Pharmacology, University of Turku, Turku, Finland.
Scheinin
Mika
M
Pohjanoksa
Katariina
K
Kallio
Jaana
J
D058647:Adrenergic alpha-2 Receptor Agonists; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D020927:Dexmedetomidine / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007150:Immunohistochemistry; D009638:Norepinephrine / Q000494:pharmacology; D016716:PC12 Cells; D020033:Protein Isoforms / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism*; D013395:Sucrose / Q000494:pharmacology; D014162:Transfection
D058647:Adrenergic alpha-2 Receptor Agonists; D000316:Adrenergic alpha-Agonists; D020033:Protein Isoforms; D018341:Receptors, Adrenergic, alpha-2; D013395:Sucrose; D020927:Dexmedetomidine; D009638:Norepinephrine