Chimeric melatonin mt(1) and melatonin-related receptors - Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt(1) receptor
S. Conway et al., Chimeric melatonin mt(1) and melatonin-related receptors - Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt(1) receptor, J BIOL CHEM, 275(27), 2000, pp. 20602-20609
Melatonin receptors bind and become activated by melatonin. The melatonin-r
elated receptor, despite sharing considerable amino acid sequence identity
with melatonin receptors, does not bind melatonin and is currently an orpha
n G protein-coupled receptor. To investigate the structure and function of
both receptors, we engineered a series of 14 chimeric receptor constructs,
allowing us to determine the relative contribution of each transmembrane do
main to Ligand binding and receptor function. Results identified that when
sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin
mt, receptor were replaced by the corresponding domains of the melatonin-re
lated receptor, the resultant chimeric receptors all displayed specific 2-[
I-125]iodomelatonin binding, Replacement of sequences incorporating transme
mbrane domains 4 or 6, however, resulted in chimeric receptors that display
ed no detectable 2-[I-125]iodomelatonin binding. The subsequent testing of
a "reverse" chimeric receptor in which sequences encoding transmembrane dom
ains 4 and 6 of the melatonin-related receptor were replaced by the corresp
onding melatonin mt, receptor sequences identified specific 2-[I-125]iodome
latonin binding and melatonin-mediated modulation of cyclic AMP levels. To
further investigate these findings, site-directed mutagenesis was performed
on residues within transmembrane domain 6 of the melatonin mt, receptor. T
his identified Gly(258) (Gly(6.55)) as a critical residue required for high
affinity ligand binding and receptor function.