Cloning of a C-terminally truncated NK-1 receptor from guinea-pig nervous system

• Published in: Brain research. Molecular brain research. 2003-03, Vol.111 (1), p.136-147
• Main Authors: Baker, Sarah J., Morris, Judy L., Gibbins, Ian L.
• Format: Article
• Language: English
• Subjects: Alternative Splicing - genetics; Amino Acid Sequence - genetics; Animals; Base Sequence - genetics; Biological and medical sciences; Cell receptors; Cell structures and functions; Central Nervous System - cytology; Central Nervous System - metabolism; Cloning, Molecular; Dendrites - metabolism; Dendrites - ultrastructure; DNA, Complementary - genetics; DNA, Complementary - isolation & purification; Female; Fluorescent Antibody Technique; Fundamental and applied biological sciences. Psychology; Ganglia, Sympathetic - cytology; Ganglia, Sympathetic - metabolism; Guinea Pigs; Male; Microscopy, Confocal; Microtubule-Associated Proteins - metabolism; Molecular and cellular biology; Molecular Sequence Data; Mutation - genetics; Neurons - cytology; Neurons - metabolism; Neuropeptide receptors; Peripheral Nervous System - cytology; Peripheral Nervous System - metabolism; Protein Isoforms - genetics; Protein Isoforms - isolation & purification; Protein Structure, Tertiary - genetics; Receptor; Receptors, Neurokinin-1 - genetics; Receptors, Neurokinin-1 - isolation & purification; RNA, Messenger - metabolism; RT-PCR; Spinal cord; Splice-variant; Substance P; Sympathetic
• ISSN: 0169-328X; 1872-6941
• DOI: 10.1016/S0169-328X(03)00002-0

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.