Multiple tachykinins are produced and secreted upon post-translational processing of the three substance P precursor proteins, alpha-, beta-, and gamma-preprotachykinin. Expression of the preprotachykinins in AtT-20 cells infected with vaccinia virus recombinants.
The rat preprotachykinin I gene mRNA is alternatively spliced to yield three different mRNA species differing in their protein coding regions. We have produced recombinant vaccinia viruses expressing alpha-, beta-, and gamma-preprotachykinin to examine the tachykinin-related peptides produced upon post-translational processing of each individual precursor. Infection of BSC-40 or AtT-20 cell lines with a beta-preprotachykinin-encoding vaccinia virus recombinant results in the expression of the precursor protein. The pro-form (signal peptide removed) can be immunoprecipitated from extracts of infected cells. Infected cells of both types secrete into the culture medium a product(s) which reacts in radioimmunoassay with an antiserum shown to recognize precursor as well as mature substance P. Infected AtT-20, but not BSC-40, cells secrete into the culture medium a processed form(s) of beta-preprotachykinin which reacts in radioimmunoassay with an anti-serum which recognizes the amidated carboxyl terminus of substance P. The molecular nature of the tachykinin products produced in and secreted from AtT-20 cells infected with alpha-, beta-, and gamma-preprotachykinin-encoding recombinants was analyzed by combined high performance liquid chromatography and radioimmunoassay. Peptides were identified based on comigration with synthetic standards and antisera cross-reactivity. We determined that alpha-preprotachykinin is processed to the mature undecapeptide, substance P. beta-Preprotachykinin was processed into multiple products, including substance P, neurokinin A, neurokinin A(3-10), and neuropeptide K. gamma-Preprotachykinin was processed into substance P, neurokinin A, neurokinin A(3-10), and neuropeptide gamma. These five tachykinin peptide products were all routed through the regulated secretory pathway and were secreted into the medium in a cAMP-stimulatable fashion. Since all of these peptides have been shown to be biologically active, it is important to consider the biological consequences of their co-secretion in vivo.
Department of Anatomy and Neurobiology, Washington University School of Medicine, St Louis, Missouri 63110.
M R
MR
MacDonald
J
J
Takeda
C M
CM
Rice
J E
JE
Krause
D000818:Animals; D002460:Cell Line; D002472:Cell Transformation, Viral; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005822:Genetic Vectors; D010911:Pituitary Neoplasms; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D011863:Radioimmunoassay; D015320:Tachykinins / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics*
D011498:Protein Precursors; D015320:Tachykinins; C044206:preprotachykinin; D012321:DNA-Directed RNA Polymerases