O

Melatonin

Fig. 1. Melatonin synthetic pathways (see text for details).

role of the SCN is to influence neurons of the superior cervical ganglion (SCG), which send axonal processes directly to the pineal gland. When stimulated, sympathetic SCG neurons release norepinephrine into the pineal, activating P-adrenergic receptors on the plasma membrane of pinealocytes. The receptors initiate a signaling cascade resulting in the production of cAMP, which stimulates the production of melatonin mainly by activating transcription of the NAT gene (6). Although NAT mRNA levels correlate very well with melatonin production, additional effects of cAMP on protein stability and serotonin synthesis in the pineal are under active investigation.

Fig. 2. Structure of PINA in comparison with ATP7B. (A) PINA cDNA structure. Functional domains that are conserved among copper transporters are marked as follows: copper, copper-binding domains; Td, transduction domain; Ch/Ph, channel/phosphorylation domain; ATP, ATP-binding domain. The potential copper-binding HXXM motif conserved in ATP7B proteins is also marked. The hatched region at the 5' end of PINA cDNA represents PINA-specific untranslated sequence. (B) PINA genomic structure. Transcriptional initiation site of PINA mRNA is located in the intron 8 (9) of ATP7B gene.

Fig. 2. Structure of PINA in comparison with ATP7B. (A) PINA cDNA structure. Functional domains that are conserved among copper transporters are marked as follows: copper, copper-binding domains; Td, transduction domain; Ch/Ph, channel/phosphorylation domain; ATP, ATP-binding domain. The potential copper-binding HXXM motif conserved in ATP7B proteins is also marked. The hatched region at the 5' end of PINA cDNA represents PINA-specific untranslated sequence. (B) PINA genomic structure. Transcriptional initiation site of PINA mRNA is located in the intron 8 (9) of ATP7B gene.

4. PINA AND PINEAL CIRCADIAN RHYTHMS

To understand the molecular regulation of the pineal gland at night, we attempted to identify nocturnal genes expressed in the pineal gland by performing subtractive hybridization (7). PINA was among the first of these genes to be characterized.

Sequence analysis of PINA revealed that it is an alternatively spliced form of ATP7B, the copper-transporting ATPase mutated in Wilson's disease (WD) patients (Fig. 2A). In the PINA message, sequences encoding the N-terminal half of ATP7B are replaced by a unique untranslated 300-base-pair leader sequence. The PINA protein, therefore, represents only the C-terminal half of ATP7B. The 300-base-pair leader sequence has been shown to be part of an intron immediately upstream of exon 9 of ATP7B, suggesting that PINA is driven by a novel pineal/retina-specific promoter (8) nested within the center of the ATP7B gene (9) (Fig. 2B). The PINA promoter in fact contains a single CRE and multiple PIRE sites, which may be important for cAMP inducibility and pineal/retina tissue-specific transcription, respectively (8).

Using probes unique for PINA, we determined that PINA is expressed exclusively in the pineal gland and the retina. Analysis by in situ hybridization demonstrates that PINA is expressed abundantly in selected populations of pinealocytes. In the retina, PINA is expressed in the retinal pigment epithelium and a subset of photoreceptors at night. Northern blot analysis also demonstrated dramatic temporal regulation of PINA mRNA that coincides precisely with the expression of NAT mRNA and increased melatonin production (Fig. 3). Like NAT, the expression of PINA is controlled by adrenergic stimulation of the pineal gland by the SCG (10).

5. IMPLICATIONS OF FUNCTIONAL ANALYSIS OF PINA

Pineal night-specific ATPase is predicted to encode a 665-amino-acid protein with a molecular weight of 74 kDa. The protein contains only four of eight transmembrane domains predicted for

Fig. 3. Temporal expression pattern of PINA mRNA in adult pineals. Northern blot of pineal RNA collected from rats at 2-h intervals was hybridized sequentially with full-length PINA, NAT (7), and GAPDH probes.

ATP7B (11) and retains the transduction, channel/phosphorylation, and ATPase catalytic domains. PINA does not contain five proposed metal-binding domains within the N-terminus of ATP7B, which have been postulated to bind copper and regulate activity of the transporter (Fig. 2A).

Surprisingly, despite the deletion of a bulk of the ATP7B protein, PINA functions as a copper transporter when introduced into yeast complementation assays, although its activity is much lower than the ATP7A protein analyzed as a control (10). The functional integrity of PINA provides some useful insight into the structure requirements of other ATPases. PINA's structure suggests that previously identified critical N-terminal regions of ATP7B may not be essential for function. Moreover, regions conserved in the C-terminus may take on significance of greater magnitude than previously suspected.

We have sought to explain how PINA functions without N-terminal metal-binding motifs by examining its sequence for possible alternative metal-binding motifs in the C-terminus. The MXXM motif has been previously implicated in copper binding in bacterial copper transporters, but we did not find these motifs in PINA. Instead, we identified 3 copies of a HXXM motif in the C-terminus of the protein, which is also present in 11 copper-transporting proteins from bacteria and vertebrates (10). In some bacterial proteins, the motif was repeated five times (Table 1). Based on this analysis, we suggest that the HXXM motif is involved in binding copper and that, in PINA, this is sufficient for copper transport. We further speculate that the N-terminal domain of ATP7B, missing in PINA, plays a regulatory, rather than essential, role in copper transport. Experimental verification is forthcoming.

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