Areas of Interest
Sympathetic neurons; peptides; vesicles; enzymes; tissue
culture; development; pituitary.
Nearly every neuron in the brain secretes a biologically
active peptide at its target cells along with one or more
conventional neurotransmitters; many neurons secrete several
bioactive peptides. Diversity in bioactive peptides is created
by the existence of multiple closely related gene products
encoding similar but not identical peptides, and by distinct
patterns in post-translational processing of the propeptide
precursors, so that a single peptide precursor gives rise to
distinct peptide products in distinct tissues. The enzymes
involved in peptide processing, and questions of how the peptide
precursors and enzymes manage to find each other, to get
together in the same secretory granules, are the focus of this
lab group. We use a number of immortal cell lines in culture
which express an endocrine phenotype, and we culture primary
neurons and endocrine cells which behave in culture as well as
the cells which remain in the intact animal.
The cultured cells allow us to manipulate the environment and
the genes expressed by the cells, often in ways impossible in
the whole animal. We transfect cells to introduce or knock out
genes of interest, or we use viral vectors to infect primary
cells and force them to make proteins that nature never
intended. We also produce, in large quantities in fibroblast
cell lines and bacteria, the proteins we express in smaller
amounts in neurons and endocrine cells, to enable detailed
enzymology to be performed. Questions of routing of the proteins
to secretory granules -- why do all those molecules end up in
the same secretory granule, anyway, to be stored for days after
the few hours crucial to biosynthetic processing? -- are also
examined, using transfections, infections, and yeast two-hybrid
interaction systems.
Lab Rotation Projects
Project: Expressing Novel Forms of Trio.
Trio is one of the 2 members of the Kalirin-Trio family of
multifunctional GEFs (guanine nucleotide exchange factors) with
spectrin, SH3 and kinase domains. Kalirin and Trio are present
in most/all neurons and are likely to play many roles in axon
guidance, dendritic maintenance, synapse formation. Trio cDNAs
will be built into mammalian expression vectors and expressed in
fibroblasts, pituitary cells and neurons. Molecular biology,
cell culture, immunocytochemistry, Western analyses.
Project: Effects of estrogens on hippocampal neurons (dendritic
spines).
Estrogens have remarkable effects on dendritic spines of neurons
in several regions of the brain, perhaps most notably the
hippocampus. Primary cultures (slice and dissociated cells) will
be prepared and treated with estrogens and estrogen antagonists
to investigate the control of dendritic spine morphology and
expression of Kalirin. Kalirin is one of the 2 members of the
Kalirin-Trio family of multifunctional GEFs (guanine nucleotide
exchange factors) with spectrin, SH3 and kinase domains; Kalirin
is known to be required for maintenance of normal dendritic
spines. Cell culture, immunocytochemistry, Western analyses.
Project: P-CIP2; a protein kinase with an important role
in peptide secretion?
PAM is the large dense core vesicle integral membrane protein
which amidates bioactive peptides; PAM intracellular trafficking
depends on its cytosolic tail. P-CIP2 is a protein kinase
identified as an interactor with the cytosolic tail of PAM. New
antisera to PCIP2 will be raised and characterized. Antisense
and siRNA techniques will be used to assess the levels of
endogenous P-CIP2 in neurons and pituitary cells;
over-expression of P-CIP2 and inactive P-CIP2 will be used to
monitor effects on the intracellular routing and secretion of
peptides and peptide processing enzymes. Cell culture, Western
analyses, transfection techniques, ELISAs.
Publications
Selected Publications
McPherson CE, Eipper BA, Mains RE. Kalirin expression is
regulated by multiple promoters. J Mol Neurosci 22: 109-120,
2003.
Ma XM, Huang J, Wang Y, Eipper BA, Mains RE, Kalirin. A
multifunctional rho Guanine nucleotide exchange factor, is
necessary for maintenance of hippocampal pyramidal neuron
dendrites and dendritic spines. The Journal of Neuroscience: the
Official Journal of the Society for Neuroscience, 23(33),
10593-603, Nov. 2003.
Chei FY, Eipper BA, Mains RE, Fricker LD. Quantitative
peptidomics of pituitary glands from mice deficient in copper
transport. Cellular and Molecular Biology (noisy-le-grand,
France), 49(5), 713-22, Jul. 2003.
Bell J, El Meskini R, D'Amato D, Mains RE, Eipper BA.
Mechanistic investigation of peptidylglycine alpha-hydroxylating
monooxygenase via intrinsic tryptophan fluorescence and
mutagenesis. Biochemistry, 42(23), 7133-42, Jun. 2003.
El Meskini R, Culotta VC, Mains RE, Eipper BA. Supplying
copper to the cuproenzyme peptidylglycine alpha-amidating
monooxygenase. Journal of Biological Chemistry, 278(14),
12278-84, Apr. 2003.
Steveson TC, Ciccotosto GD, Ma XM, Mueller GP, Mains RE,
Eipper BA. Menkes protein contributes to the function of
peptidylglycine alpha-amidating monooxygenase. Endocrinology,
144(1), 188-200, Jan. 2003.
Penzes P, Beeser A, Chernoff J, Schiller MR, Eipper BA, Mains
RE, Huganir RL. Rapid induction of dendritic spine morphogenesis
by trans-synaptic ephrinB-EphB receptor activation of the
Rho-GEF kalirin. Neuron, 37(2), 263-74, Jan. 2003.
Marx R, Mains RE. Routing of membrane proteins to large dense
core vesicles in PC12 cells. J Mol Neurosci 18(1-2), 113-27,
2002.
Jaron S, Mains RE, Eipper BA, Blackburn NJ. The catalytic
role of the copper ligand H172 of peptidylglycine alpha-hydroxylating
monooxygenase (PHM): a spectroscopic study of the H172A mutant.
Biochemistry, 41(44), 13274-82, Nov. 2002.
Kolhekar AS, Bell J, Shiozaki EN, Jin L, Keutmann HT, Hand
TA, Mains RE, Eipper BA. Essential features of the catalytic
core of peptidyl-alpha-hydroxyglycine alpha-amidating lyase.
Biochemistry, 41(41), 12384-94, Oct. 2002.
May V, Schiller MR, Eipper BA, Mains RE. Kalirin Dbl-Homology
Guanine Nucleotide Exchange Factor 1 Domain Initiates New Axon
Outgrowths via RhoG-Mediated Mechanisms. The Journal of
Neuroscience : the Official Journal of the Society for
Neuroscience, 22(16), 6980-90, Aug. 2002.
Ma XM, Mains RE, Eipper BA. Plasticity in hippocampal
peptidergic systems induced by repeated electroconvulsive shock.
Neuropsychopharmacology : Official Publication of the American
College of Neuropsychopharmacology, 27(1), 55-71, Jul. 2002.
Bruzzaniti A, Mains RE. Enzymatic activity of soluble and
membrane tethered peptide pro-hormone convertase 1. Peptides,
23(5), 863-75, May 2002.
McPherson CE, Eipper BA, Mains RE. Genomic organization and
differential expression of Kalirin isoforms. Gene, 284(1-2),
41-51, Feb. 2002.
 View more publications, see
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