Cloning of a C-terminally truncated NK-1 receptor from guinea-pig nervous system.
In order to examine the possibility that some actions of substance P may be mediated by a variant of the neurokinin-1 (NK-1) receptor, we isolated and sequenced the cDNA encoding a truncated NK-1 receptor from guinea-pig celiac ganglion and brain mRNA by two-step RT-PCR based on the 3'RACE method. The truncated NK-1 receptor sequence corresponded to a splice variant missing the final exon 5, and encoded a 311-amino acid protein that was truncated just after transmembrane domain 7, in an identical position to a truncated variant of the human NK-1 receptor. Thus, the truncated NK-1 receptor lacked the intracellular C-terminus sequence required for the phosphorylation and internalisation of the full-length NK-1 receptor. Using a sensitive one-step semi-quantitative RT-PCR assay, we detected mRNA for both the full length and truncated NK-1 receptors throughout the brain, spinal cord, sensory and autonomic ganglia, and viscera. Truncated NK-1 receptor mRNA was present in lower quantities than mRNA for the full-length NK-1R in all tissues. Highest levels of mRNA for the truncated NK-1 receptor were detected in coeliac ganglion, spinal cord, basal ganglia and hypothalamus. An antiserum to the N-terminus of the NK-1 receptor labelled dendrites of coeliac ganglion neurons that were not labelled with antisera to the C-terminus of the full length NK-1 receptor. These results show that a C-terminally truncated variant of the NK-1 receptor is likely to be widespread in central and peripheral nervous tissue. We predict that this receptor will mediate actions of substance P on neurons where immunohistochemical evidence for a full-length NK-1 receptor is lacking.
Baker
Sarah J
SJ
Department of Anatomy and Histology, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia.
Morris
Judy L
JL
Gibbins
Ian L
IL
D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002490:Central Nervous System / Q000166:cytology / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D003712:Dendrites / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D005455:Fluorescent Antibody Technique; D005728:Ganglia, Sympathetic / Q000166:cytology / Q000378:metabolism; D006168:Guinea Pigs; D008297:Male; D018613:Microscopy, Confocal; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D017933:Peripheral Nervous System / Q000166:cytology / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification; D017434:Protein Structure, Tertiary / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000235:genetics / Q000302:isolation & purification*
D018076:DNA, Complementary; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D018040:Receptors, Neurokinin-1