Problems in N-terminal tagging

[Tarja Kokkola]

Dear fellow bio-scientists,

Is it necessary to add any signal peptide sequences or a Kozak
sequence when
building a N-terminally tagged G protein-coupled receptor construct?
By now,
I have always just added a tag sequence after the initiation codon of
my
receptor and have had success in expressing the tagged receptor in
mammalian
cells. There has been no consensus Kozak nucleotide sequence before
the
initiation codon, either.

I heard that membrane proteins need a N-terminal signal peptide which
guides
them to the plasma membrane and that a N-terminal tag would be cut off
if it had been inserted before that peptide (because the signal
peptide is cleaved off during the transport process). Can this be true
for some GPCRs??

Please help me clarify that problem. Some Internet links regarding
N-terminal signal sequences would be welcome, too.

Thanks,
Tarja Kokkola