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 microM 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.
Vollmer
J Y
JY
Département Récepteurs et Protéines Membranaires, CNRS UPR 9050, Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, 67400 Illkirch, France.
Alix
P
P
Chollet
A
A
Takeda
K
K
Galzi
J L
JL
D000818:Animals; D001896:Boron Compounds; D002118:Calcium / Q000378:metabolism; D002451:Cell Compartmentation; D002460:Cell Line; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D006801:Humans; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D051381:Rats; D018041:Receptors, Neurokinin-2 / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions / Q000378:metabolism*
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D008024:Ligands; D018041:Receptors, Neurokinin-2; D011993:Recombinant Fusion Proteins; D002118:Calcium