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Faculty
Richard Mains
Professor of Neuroscience
mains@nso.uchc.edu
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.
Mains Lab Page
Additional Information
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. |
