Functional interactions between native Gs-coupled 5-HT receptors in HEK-293 cells and the heterologously expressed serotonin transporter.
In HEK-293 cells, serotonin (5-hydroxytryptamine, 5-HT) was found to induce cAMP production showing pharmacological characteristics consistent with the 5-HT(7) receptor. The presence of 5-HT(7) (and 5-HT(6)) receptor mRNA was confirmed by RT-PCR. Stable HEK-293 cell lines expressing either wild-type or haemagglutinin (HA)-tagged human 5-HT transporter (SERT) were selected and SERT function was confirmed using [3H]5-HT transport. The presence of SERT caused a 10-fold reduction in the potency of 5-HT-induced cAMP production compared to control cells. Downstream signalling by 5-HT(6/7) receptors could be detected as 5-HT-induced protein kinase A activation and phosphorylation of MAP kinase and CREB using phospho-specific antibodies. SERT inhibitors reversed the reduction in potency of 5-HT-induced cAMP production caused by the presence of SERT, resulting in a concentration-dependent left shift in EC(50) values but also a progressive decrease in the maximal response. Thus, when antidepressants were used to block SERT activity, 5-HT receptor signalling was effectively clamped within a mid-range.
Johnson
Melanie S
MS
MRC Membrane and Adapter Proteins Co-operative Group, School of Biomedical and Clinical Laboratory Sciences, Edinburgh University, George Square, Edinburgh EH8 9XD, UK. M.S.Johnson@ed.ac.uk
Lutz
Eve M
EM
Firbank
Susan
S
Holland
Pamela J
PJ
Mitchell
Rory
R
D000928:Antidepressive Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000187:drug effects / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D019950:Mitogen-Activated Protein Kinase 1 / Q000187:drug effects / Q000378:metabolism; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism; D011985:Receptors, Serotonin / Q000187:drug effects / Q000235:genetics*; D012701:Serotonin / Q000378:metabolism* / Q000494:pharmacology; D050486:Serotonin Plasma Membrane Transport Proteins; D009435:Synaptic Transmission / Q000187:drug effects / Q000235:genetics
D000928:Antidepressive Agents; D002352:Carrier Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011985:Receptors, Serotonin; C493230:SLC6A4 protein, human; D050486:Serotonin Plasma Membrane Transport Proteins; C097216:serotonin 6 receptor; C104425:serotonin 7 receptor; D012701:Serotonin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D019205:GTP-Binding Protein alpha Subunits, Gs