Two distinct pathways for histamine H2 receptor down-regulation. H2 Leu124 --> Ala receptor mutant provides evidence for a cAMP-independent action of H2 agonists.
Pretreatment of Chinese hamster ovary cells expressing the histamine H2 receptor (CHOrH2 cells) with histamine resulted in a time-dependent (t1/2 approximately 7 h) and dose-dependent (EC50=18 nM) H2 receptor down-regulation measured as [125I]iodoaminopotentidine binding (44+/-10% down-regulation). Pretreatment of CHOrH2 cells with cholera toxin or forskolin also led to H2 receptor down-regulation. Forskolin time-dependently (t1/2 approximately 7 h) and dose-dependently (EC50 = 0.3 microM) induced H2 receptor down-regulation. Both histamine and forskolin induced rapid down-regulation of H2 receptor mRNA levels, probably caused by mRNA destabilization. Recently, Moro et al. (Moro, O. Lameh, J., Hogger, P., and Sadée, W. (1993) J. Biol. Chem. 268, 22273-22276) showed that hydrophobic amino acids in a conserved G-protein-coupled receptor motif in the second intracellular loop are implicated in G-protein coupling. To uncouple the H2 receptor from the Gs-protein, we introduced the Leu124 --> Ala mutation in the second intracellular loop of the H2 receptor. The H2 Leu124 --> Ala mutant showed altered agonist-binding parameters, attenuated histamine-induced cAMP production, and was down-regulated by concentrations of histamine that did not give rise to cAMP production. Taken together, in CHOrH2 cells, H2 receptor down-regulation appears to be induced by two distinct pathways, a cAMP-dependent and cAMP-independent pathway.
Smit
M J
MJ
Leiden/Amsterdam Center for Drug Research, Department of Pharmacochemistry, Vrije Universiteit, Amsterdam, The Netherlands.
Roovers
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Timmerman
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H
van de Vrede
Y
Y
Alewijnse
A E
AE
Leurs
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R
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology*; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017931:DNA Primers; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D015870:Gene Expression / Q000187:drug effects*; D006146:Guanidines / Q000378:metabolism; D006632:Histamine / Q000494:pharmacology*; D017442:Histamine Agonists / Q000494:pharmacology*; D006635:Histamine H2 Antagonists / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D011970:Receptors, Histamine H2 / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D006146:Guanidines; D017442:Histamine Agonists; D006635:Histamine H2 Antagonists; D007457:Iodine Radioisotopes; D012333:RNA, Messenger; D011970:Receptors, Histamine H2; D011994:Recombinant Proteins; C063279:iodoaminopotentidine; D005576:Colforsin; D006632:Histamine; D002772:Cholera Toxin; D000242:Cyclic AMP; D015056:1-Methyl-3-isobutylxanthine