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The neuroendocrine polypeptide 7B2 is a precursor protein.
Ayoubi TA
,
van Duijnhoven HL
,
van de Ven WJ
,
Jenks BG
,
Roubos EW
,
Martens GJ
.
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The neuroendocrine protein 7B2 is highly conserved and widely present in neurons and endocrine cells. It is coexpressed with the prohormone proopiomelanocortin (POMC) in the intermediate lobe of the pituitary gland of Xenopus laevis. To study the biosynthesis of 7B2 in this amphibian, an anti-7B2 monoclonal antibody was used in immunoprecipitation analysis of newly synthesized radiolabeled proteins, produced by pulse and pulse-chase-incubated neurointermediate lobes. Following a 15-min pulse incubation, a single immunoprecipitable protein of 25 kDa was synthesized. During subsequent chase incubation, this newly synthesized 7B2 protein was processed to an 18-kDa immunoprecipitable form. Analysis of the chase incubation medium revealed that only the 18-kDa processed product of 7B2, and not 7B2 itself, had been secreted. This secretion is a regulated process because it was blocked completely by the dopamine receptor agonist apomorphine. A study of protein biosynthesis in lobes treated with tunicamycin to prevent N-linked glycosylation showed that in contrast to POMC and an 18-kDa derivative of POMC, neither 7B2 nor its 18-kDa derivative was glycosylated. Chemical and enzymatic peptide mapping showed that processing of 7B2 occurs in the carboxyl-terminal region. The function of the 7B2 protein is unknown; the present results show that 7B2 itself is a precursor molecule and can only have an intracellular function whereas an extracellular function can only be attributed to 7B2-derived peptides.
FIG. 1. Pulse-chase and immunoprecipitation analysis. Radiolabeled
proteins produced by pulse and pulse-chase-incubated
neurointermediate lobes of X. laeuis (four lobes/lane) were immunoprecipitated
using normal mouse serum (lane 1) or anti-7B2 monoclonal
antibody MON-100 (lanes 2-8). Lanes 1-6 represent immunoprecipitated
lobe extracts, and lanes 7 and 8 are immunoprecipitated
chase media. Lanes 1 and 2, 15-min pulse; lane 3, 30-min pulse;
lane 4, 60-min pulse; lane 5, 60-min pulse and 2-h chase; lane 6, 60-
min pulse and 4-h chase; lane 7, as lane 5; lane 8, as lane 6. Lanes 9-
13, total extracts of labeled proteins (0.2 lobe/lane) corresponding to
lanes 2-6.
FIG. 2. Effect of apomorphine on the secretion of newly
synthesized 7B2. Lobes were pulsed for 90 min and chased for 4 h
with (lanes 1, 3, and 5) or without 10mG M apomorphine (lanes 2, 4,
and 6) and subjected to immunoprecipitation analysis. Four lobes
were used per lane. Lanes 1 and 2, immunoprecipitated lobe extracts;
lanes 3 and 4, immunoprecipitated incubation media; lanes 5 and 6,
total extracts of labeled proteins (0.2 lobe/lane).
FIG. 3. Effect of tunicamycin on newly synthesized 7B2.
Lobes were preincubated for 16 h with (lanes 1, 3, and 5) or without
(lanes 2,4, and 6) 10 rg/ml tunicamycin, pulse incubated for 3 h, and
subjected to immunoprecipitation analysis. Four lobes were used per
lane. Lanes 1 and 2, immunoprecipitated lobe extracts; lanes 3 and 4,
immunoprecipitated incubation media; lanes 5 and 6, total extracts
of labeled proteins (0.2 lobe/lane). Lanes I, 3, and 5 are tunicamycin
treated; lanes 2, 4, and 6 are controls. *, not glycosylated POMC.
FIG. 4. Cyanogen bromide peptide mapping of immunoprecipitated
7B2. After a 5-h pulse incubation, radiolabeled 7B2 was
immunoprecipitated from the incubation medium (lanes I and 2) or
from the lobe extracts (lanes 3 and 4). Lanes I and 3 represent
control-incubated, immunoprecipitated, radiolabeled 7B2; lanes 2 and
4 represent immunoprecipitated, radiolabeled 7B2 cleaved by 10%
cyanogen bromide.
FIG. 5. Scheme of VS protease digestion of 7B2 and its l&3-
kDa product. Small arrow denote glutamate residues at which
cleavage occurs. Asterisks indicate [“Hjtryptophan residue.
FIG. 6. Reversed-phase HPLC analysis of VS digests of 7B2
and its 1%kDa product. Lobes were pulse labeled for 3 h with [“HI
tryptophan. Newly synthesized 7B2 and the 18-kDa product were
immunoprecipitated, separated by SDS-polyacrylamide gel electrophoresis,
cut out from the gel, and digested with V8 protease. Shown
are HPLC profiles of Vb-digested A, 25-kDa precursor from lobe
extracts; E, 18kDa product from lobe extracts; and C, 18-kDa product
from the incubation medium. The profiles are representative data of
three separate experiments.
