cAMP-Mediated Stimulation of Tyrosine Hydroxylase mRNA Translation Is Mediated by Polypyrimidine-Rich Sequences within Its 3′-Untranslated Region and Poly(C)-Binding Protein 2

Abstract

Tyrosine hydroxylase (TH) plays a critical role in maintaining the appropriate concentrations of catecholamine neurotransmitters in brain and periphery, particularly during long-term stress, long-term drug treatment, or neurodegenerative diseases. Its expression is controlled by both transcriptional and post-transcriptional mechanisms. In a previous report, we showed that treatment of rat midbrain slice explant cultures or mouse MN9D cells with cAMP analog or forskolin leads to induction of TH protein without concomitant induction of TH mRNA. We further showed that cAMP activates mechanisms that regulate TH mRNA translation via cis-acting sequences within its 3Ј-untranslated region (UTR). In the present report, we extend these studies to show that MN9D cytoplasmic proteins bind to the same TH mRNA 3Ј-UTR domain that is required for the cAMP response. RNase T1 mapping demonstrates binding of proteins to a 27-nucleotide polypyrimidine-rich sequence within this domain. A specific mutation within the polypyrimidine-rich sequence inhibits protein binding and cAMP-mediated translational activation. UV-cross-linking studies identify a ϳ44-kDa protein as a major TH mRNA 3Ј-UTR binding factor, and cAMP induces the 40-to 42-kDa poly(C)-binding protein-2 (PCBP2) in MN9D cells. We show that PCBP2 binds to the TH mRNA 3Ј-UTR domain that participates in the cAMP response. Overexpression of PCBP2 induces TH protein without concomitant induction of TH mRNA. These results support a model in which cAMP induces PCBP2, leading to increased interaction with its cognate polypyrimidine binding site in the TH mRNA 3Ј-UTR. This increased interaction presumably plays a role in the activation of TH mRNA translation by cAMP in dopaminergic neurons.Tyrosine hydroxylase (TH) gene expression is tightly controlled by both transcriptional and post-transcriptional mechanisms (Kumer and Vrana, 1996;Sabban and Kvetnanský , 2001;Wong and Tank, 2007). The appropriate synthesis of dopamine, norepinephrine, and epinephrine is highly dependent on TH activity; hence, these control mechanisms play an essential role in the homeostatic regulation of catecholamine levels in both brain and periphery. Transcriptional regulation of the TH gene has been highly studied. TH gene transcription is activated in adrenal medulla, locus coeruleus, and other tissues in response to many stimuli, including stress, hypoxia, and treatment with cholinergic drugs or reserpine. If this activation is sustained (1-2 h), then TH mRNA is induced. The TH gene proximal promoter comprises numerous well-studied response elements, many of which participate in this transcriptional response.Even though less well studied, post-transcriptional mechanisms also influence TH expression in adrenal medulla and brain. A number of in vivo studies have measured discrepancies between changes in TH gene transcription rate and TH mRNA levels in response to different stressors or drug treatments, suggesting that TH mRNA stability is regulated (Chang et al., 2000;Sun et al., 2004;Osterhout et a...

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