HUMAN NOREPINEPHRINE TRANSPORTER - BIOSYNTHETIC-STUDIES USING A SITE-DIRECTED POLYCLONAL ANTIBODY

Antibodies have been raised against synthetic peptides derived from th e predicted primary sequence of the human cocaine and antidepressant-s ensitive norepinephrine (NE) transporter (NET). One antibody (N430), r aised and purified against a putative intracellular human norepinephri ne transporter (hNET) epitope, detects hNET expression in a stably tra nsfected cell line (LLC-NET) by indirect immunofluorescence only in th e presence of detergent, while no immunoreactivity is observed in eith er the parental cells (LLC-PK1) or in LLC-NET cells incubated with pre immune sera or peptide absorbed antibody. N430 immunoblots of LLC-NET cell extracts reveal two major immunoreactive hNET species in these ce lls, migrating at 80 and 54 kDa, respectively. Pulse-chase N430 immuno precipitation studies confirm that the 54-kDa species is a transient, glycosylated intermediate of a longer lived, more highly glycosylated protein with an apparant M(r) of 80,000. In contrast, a 54-kDa species is the primary hNET product in vaccinia virus T7-infected HeLa cells, transiently transfected with hNET cDNA PNGase F digestion of extracts prepared from LLC-NET- and hNET-transfected HeLa cells convert all im munoreactive species to a 46-kDa form, equivalent to that observed fol lowing incubation of whole cells with the glycosylation inhibitor tuni camycin. As transiently transfected HeLa and stable LLC-NET cells exhi bit a pharmacologically similar NE transport activity, it appears like ly that the additional glycosylation evident in the stable line does n ot contribute significantly to antagonist sensitivity. On the other ha nd, NE transport and antagonist ([I-125]RTI-55) binding assays on whol e LLC-NET cells treated with tunicamycin reveal a pronounced reduction in NE transport activity and hNET membrane density paralleled by an i nability of NET proteins to replenish the higher M(r) hNET pool. These findings suggest an obligate role for N-linked glycosylation in hNET biosynthetic maturation, stability, and functional expression. In summ ary, N430 antibody is a useful tool for the visualization and characte rization of hNET gene products and has permitted the first direct eval uation of biosynthetic steps leading to functional catecholamine trans porter expression.