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The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney-derived COS-7, COS-1 and HEK-293 but not in other mammalian cell lines (CHO, Y1, Ltk-), we have characterized a putative functional dopamine transporter displaying a high affinity (Km approximately 250 nM) and a low capacity (approximately 0.1 pmol/10(5) cells/min) for [3H]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter. Estimated Ki values of numerous substrates and inhibitors for the COS-dopamine transporter and the cloned human neuronal transporter (human dopamine transporter) correlate well with the exception of a few notable compounds, including the endogenous neurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine. As with native neuronal and cloned dopamine transporters, the uptake velocity was sodium-sensitive and reduced by phorbol ester pre-treatment. Two mRNA species of 3.8 and 4.0 kb in COS-7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue. A reverse-transcribed PCR analysis confirmed the existence of a processed dopamine transporter. However, no immunoreactive proteins of expected dopamine transporter molecular size or [3H]WIN 35,428 binding activity were detected. A partial cDNA of 1.3 kb, isolated from a COS-1 cDNA library and encoding transmembrane domains 1-6, displayed a deduced amino acid sequence homology of approximately 96% to the human dopamine transporter. Taken together, the data suggest the existence of a non-neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter.

The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney-derived COS-7, COS-1 and HEK-293 but not in other mammalian cell lines (CHO, Y1, Ltk−), we have characterized a putative functional dopamine transporter displaying a high affinity (Km∼250 nM) and a low capacity (∼0.1 pmol/105 cells/min) for [3H]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter. Estimated Ki values of numerous substrates and inhibitors for the COS-dopamine transporter and the cloned human neuronal transporter (human dopamine transporter) correlate well with the exception of a few notable compounds, including the endogenous neurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine. As with native neuronal and cloned dopamine transporters, the uptake velocity was sodium-sensitive and reduced by phorbol ester pre-treatment. Two mRNA species of 3.8 and 4.0 kb in COS-7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue. A reverse-transcribed PCR analysis confirmed the existence of a processed dopamine transporter. However, no immunoreactive proteins of expected dopamine transporter molecular size or [3H]WIN 35,428 binding activity were detected. A partial cDNA of ∼1.3 kb, isolated from a COS-1 cDNA library and encoding transmembrane domains 1–6, displayed a deduced amino acid sequence homology of ∼96% to the human dopamine transporter. Taken together, the data suggest the existence of a non-neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter.

An approach based on homology probing was used to clone a partial cDNA encoding a novel melatonin (ML) receptor (MLR) from chicken (Gallus domestlcus) brain. Based on availa- ble deduced amino-acid sequence, the chicken MLR (cMLR) displayed greater sequence homology to the frog (Xcnopus) MLR than cloned human/mammalian receptors, with overall identities of 73% and 66%, respectively. In order to gain functional expres- sion, a chimeric frog/chicken (flc)MLR was constructed in which the 5' end of the cMLR, including the N-terminus, TMI and part of the first intracellular loop was substituted by fMLR sequence. (12Sl)lodo-ML bound with high affinity (Ko of ~35 pM) to COS-7 cells transiently expressing the flcMLR in a saturable and gua- nine nucleotide-sensitive manner with the following rank order of potency: 2-iodo-ML > ML > 6-CI-ML > $20750 > 6-OH-ML > $20642 > $20753 > N-acetyl-SHT >> S-HT. Estimated Ki values for these compounds at the flcMLR correlated well to those obtained in native chicken brain membranes. In line with the observed structural similarity to the fMLR, the flcMLR exhibited affinities for ML, 6-CI-ML and 6-OH-ML ~10-fold lower than mammalian receptors. Functionally, opposing interac- tions between ML and dopamine receptor signal transduction pathways were observed with ML potently inhibiting dopamine D1 A-receptor-mediated cAMP accumulation in cells (HEK-293) transiently co-expressing these receptors. ¢MLR mRNAs were found expressed in chicken brain and kidney with trace levels observed in the lung. The availability of cloned vertebrate MLRs distinct at both the amino acid and pharmacological level from their mammalian counterparts may now allow for the identifica- tion of those amino-acid residues and structural motifs that regu- late ML-binding specificity and affinity.