Subcellular compartmentalization of activation and desensitization of responses mediated by NK2 neurokinin receptors

A functional fluorescent neurokinin NK2 receptor was
constructed by joining enhanced green fluorescent protein
to the amino-terminal end of the rat NK2 receptor
and was expressed in human embryonic kidney cells. On
cell suspensions, the binding of fluorescent Bodipy-labeled
neurokinin A results in a saturatable and reversible
decrease of NK2 receptor fluorescence via fluorescence
resonance energy transfer. This can be quantified
for nM to mM agonist concentrations and monitored in
parallel with intracellular calcium responses. On single
cells, receptor site occupancy and local agonist concentration
can be determined in real time from the decrease
in receptor fluorescence. Simultaneous measurement of
intracellular calcium responses and agonist binding reveals
that partial receptor site occupancy is sufficient to
desensitize cellular response to a second agonist application
to the same membrane area. Subsequent stimulation
of a distal membrane area leads to a second response
to agonist, provided that it had not been exposed
to agonist during the first application. Together with
persistent translocation of fluorescent protein kinase C
to the membrane area exposed to agonist, the present
data support that not only homologous desensitization
but also heterologous desensitization of NK2 receptors
is compartmentalized to discrete membrane domains.