Functional comparison of full-length and N-terminal-truncated octopamine transporters from Lepidoptera.
We have cloned two new lepidopteran octopamine transporters (OATs), members of the solute-linked carrier family 6 (SLC6) of nutrient transporters, from the CNS of the European corn borer Ostrinia nubilalis and the cabbage white Pieris rapae. Comparison of these sequences with the previously cloned OAT from the cabbage looper Trichoplusia ni showed that the T. ni OAT sequence previously reported was truncated by 74 amino acids at the N-terminus. The cytoplasmic N-termini deduced here are considerably longer than the N-termini of other monoamine transporters in the SLC6 family and contain many more high-probability serine- and threonine-phosphorylation sites. Monoamine uptake and competitive inhibition studies on baculovirus-infected Sf9 cells expressing these three cloned OATs indicate that they are able to transport tyramine, octopamine and dopamine with high affinity (K(m) and K(i) range, 0.4 microM-2.7 microM) and capacity ((3)H-dopamine uptake by TrnOAT, 2.5 pmol/well/min). We aimed to examine the role of the N-terminus of OAT by comparing the properties of the full-length T. ni OAT with those of the previously reported N-truncated version. Results for the new full-length T. ni OAT showed no difference in the protein's affinity for octopamine or dopamine, although at low levels of viral infection it did show slightly higher transport activity ((3)H-dopamine uptake by truncated TrnOAT, 1.5 pmol/well/min). Treatment of Sf9 cells expressing full-length or truncated TrnOAT with a variety of protein kinase activators and inhibitors, however, did not change transporter activity. Neither an intact N-terminus, nor apparently a particular phosphorylation state of this extended N-terminus, is required for OAT to transport monoamines.
Donly
Cam
C
Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario, Canada. doinlyc@agr.gc.ca
Verellen
LouAnn
L
Cladman
Wendy
W
Caveney
Stan
S
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007915:Lepidoptera / Q000235:genetics* / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009655:Octopamine / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D019476:Insect Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D009655:Octopamine