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J. Biol. Chem., Vol. 276, Issue 32, 29854-29863, August 10, 2001
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From the Department of Neuroscience, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205
A key feature of the regulated secretory pathway
in neuroendocrine cells is lumenal pH, which decreases between
trans-Golgi network and mature secretory granules. Because
peptidylglycine
Response of an Integral Granule Membrane Protein to Changes
in pH*
,
-amidating monooxygenase (PAM) is one of the few
membrane-spanning proteins concentrated in secretory granules and is a
known effector of regulated secretion, we examined its sensitivity to
pH. Based on antibody binding experiments, the noncatalytic linker
regions between the two enzymatic domains of PAM show
pH-dependent conformational changes; these changes occur in
the presence or absence of a transmembrane domain. Integral membrane
PAM-1 solubilized from rat anterior pituitary or from transfected
AtT-20 cells aggregates reversibly at pH 5.5 while retaining enzyme
activity. Over 35% of the PAM-1 in anterior pituitary extracts
aggregates at pH 5.5, whereas only about 5% aggregates at pH 7.5. PAM-1 recovered from secretory granules and endosomes is highly
responsive to low pH-induced aggregation, whereas PAM-1 recovered from
a light, intracellular recycling compartment is not. Mutagenesis
studies indicate that a transmembrane domain is necessary but not
sufficient for low pH-induced aggregation and reveal a short lumenal,
juxtamembrane segment that also contributes to pH-dependent
aggregation. Taken together, these results demonstrate that several
properties of membrane PAM serve as indicators of granule pH in
neuroendocrine cells.
*
This work was supported by Grants DK32948, DK32949, and
DK09942 from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Center for Experimental Therapeutics, University
of Pennsylvania School of Medicine, Philadelphia, PA 19104.
§
Present address: Dept. of Neuroscience, University of Connecticut
Health Center, 263 Farmington Ave., Farmington, CT 06030-3401.
¶
To whom correspondence should be addressed: Dept. of
Neuroscience, MC3401, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030-3401. Tel.: 860-679-8894;
Fax: 860-679-1060; E-mail: mains@uchc.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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