Single amino acid substitutions in recombinant bovine prolactin that markedly reduce its mitogenic activity in Nb2 cell cultures.
Three amino acid residues of bovine PRL (bPRL) have been examined for their roles in the mitogenic activity of the hormone in Nb2 lymphoma cell cultures. The residues of interest, R21, R177, and K187, are conserved in eight pituitary PRLs, but not in the related, nonlactogenic bGH. Using site-specific mutagenesis, a number of recombinant methionyl bPRL variants have been prepared, each of which contained a single amino acid substitution of one of the three residues; a variety of amino acids was used for substitution. Twelve exchanges of R177 (to A, L, N, K, D, E, Y, G, S, Q, H, and F) all led to marked decreases in mitogenic activity. Even the conservative change, R177K, led to a decrease in mitogenic activity of about 90%; all the other R177 substitutions led to even more marked decreases; there was essentially complete loss of activity when the positively charged R177 was replaced by the negatively charged aspartate. Exchanges of R21 (to A, L, N, and K) were less dramatic, with the greatest decrease (79%) occurring in the case of R21A. Exchanges of K187 (to A, L, N, and R) had a relatively minor effect on the mitogenic activity of the hormone. Residues R21 and R177 in bPRL are located in putative helices 1 and 4, respectively; in the three-dimensional structure of the hormone these residues are predicted to be quite closely apposed. The results suggest that R177 and, to a lesser degree, R21 have important roles in the mitogenic activity of bPRL.
Luck
D N
DN
Department of Biochemistry, Faculty of Medicine, University of British Columbia, Vancouver, Canada.
Huyer
M
M
Gout
P W
PW
Beer
C T
CT
Smith
M
M
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000473:pathology; D004247:DNA / Q000235:genetics; D008223:Lymphoma / Q000473:pathology* / Q000503:physiopathology; D008934:Mitogens / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011388:Prolactin / Q000737:chemistry* / Q000494:pharmacology*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology
D000596:Amino Acids; D008934:Mitogens; D011994:Recombinant Proteins; D011388:Prolactin; D004247:DNA