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<p>Monoamines were first discovered at the end of the 19th century when William Bates
identified epinephrine (EPI) and noted its hemostatic effects. During the 20th century,
norepinephrine (NE), dopamine (DA), and serotonin (5HT) were discovered in both the
periphery and the brain. Due, in part, to the implication of monoamines in the etiology
of a wide range of dysfunctions, the examination of their physiological functions
became the subject of a considerable volume of research. Much progress has been made
in describing the function and endogenous regulation of these systems, as well as
their response to pharmacological intervention. However, many aspects of these systems
remain unexplored. For example, though the role of pharmacological agents in regulating
monoamine transporter function has been widely studied, relatively little is known
about basal regulation in terms of protein processing and targeting. Similarly, the
role of phosphorylation has been well characterized in the regulation of tyrosine
hydroxylase (TH), but little is known about the regulation of the closely related
tryptophan hydroxylases. The recent discovery of the second isoform of tryptophan
hydroxylase (TPH2) has brought renewed interest to this field as the majority of this
second isoform is centrally expressed and it contains an additional 41 amino acids
at the N-terminus compared to TPH1, the peripheral enzyme. To increase the understanding
of these aspects of monoamine signaling, this study characterizes the regulatory role
played by the extended N-terminus of TPH2 using mutagenesis and cell culture systems
and identifies determinants of monoamine transporter targeting and processing using
the dopamine transporter (DAT) as a model. In chapter 2, we demonstrate that TPH2
is synthesized less efficiently and is also less stable than TPH1 when expressed in
cultured cells. Furthermore, we identify a region centered upon amino acids 10-20
in TPH2 that appears responsible for the bulk of this difference. We also demonstrate
here that phosphorylation of S19 in TPH2 results in increased TPH2 stability, and
a consequent increase in 5HT production. Because this domain is unique to TPH2, these
data provide evidence for selective regulation of brain 5HT synthesis. Based on measured
uptake capacity and both visual and biochemical markers of protein localization, the
results presented in chapter 3 suggest that a conserved YAAY motif in the C-tail of
the monoamine transporters is essential for normal levels of membrane expression.
We also demonstrate that disruption of this sequence interferes to some extent with
the previously described hDAT/Hic-5 interaction. Together, the data presented here
contribute to the understanding of the physiological regulation of brain monoaminergic
signaling.</p>
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