MULTIPLE TACHYKININS ARE PRODUCED AND SECRETED UPON POST-TRANSLATIONAL PROCESSING OF THE 3 SUBSTANCE-P PRECURSOR PROTEINS, ALPHA-PREPROTACHYKININ, BETA-PREPROTACHYKININ, AND GAMMA-PREPROTACHYKININ - EXPRESSION OF THE PREPROTACHYKININS IN ATT-20 CELLS INFECTED WITH VACCINIA VIRUS RECOMBINANTS

  • 15 September 1989
    • journal article
    • research article
    • Vol. 264  (26) , 15578-15592
Abstract
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 antiserum 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 crossreactivity. 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.