Rhythmic Trafficking of TRPV2 in the Suprachiasmatic Nucleus is Regulated by Prokineticin 2 Signaling

Mice were anesthetized with xylazine and ketamine and transcardially perfused with PBS, followed by 4% paraformaldehyde in PBS. The brains were removed, post-fixed in 4% paraformaldehyde overnight, and cryoprotected with 20% sucrose in 0.1 M phosphate buffer (PB, pH 7.4) for 48 hr. The brains were subsequently frozen in isopentane and 16–20 micrometer coronal sections were cut using a cryostat. Antisense riboprobes containing the 3’ UTR of mouse PKR2 (GenBank accession number; residues 1147–2211) were generated by T7 RNA polymerase (sense probes were generated with SP6 RNA polymerase). Riboprobes was labeled with digoxigenin-UTP (DIG-UTP) (Roche) and diluted l to 50 in hybridization solution (50% formaldehyde, 10% dextran sulfate, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 0.02% BSA, 500 µg/ml tRNA, 0.3M NaCl, 10mM Tris, pH 8.0, 1mM EDTA). For the hybridization, sections were sequentially hydrated in 1x PBS (phosphate buffered saline), 0.6% HOin 1x PBS for 30 minutes, 1.2% HOin 1x PBS for 1 hr, and 0.6% HOin 1x PBS for 30 minutes. Tissue sections were fixed with 4% paraformaldehyde for 15 minutes, followed by three 1x PBS washes. Sections were pretreated with proteinase K (10 µg/ml) and pre-hybridized with the hybridization solution without probe for 1 hr at room temperature. Hybridized with the riboprobe, the sections were incubated at 60°C for 18 hours, followed by RNAase (20 µg/ml) digestion, decreasing salinity washes and a 60 minute high stringency (68°C) wash. Sections were then washed with 1x PBS and 0.2% Triton-X100 (PBST) twice, followed by a blocking solution (PBST, 3% BSA, and 5% horse serum) at room temperature for 1 hr. Sections were incubated with an anti-DIG antibody conjugated with horseradish peroxidase (Perkin Elmer) in blocking solution overnight at 4°C and washed with PBST. DIG labeled cells were revealed with the TSA Plus Fluorescence Kit from Perkin Elmer as described in the instruction manual. Sections were viewed with fluorescence microscopy prior to the immunohistochemistry. Sections were incubated with rabbit anti-TRPV2 antibody (1:1000, Santa Cruz Biotechology) for 24 hr at 4°C. After washes with PBST, the sections were incubated with the secondary antibody conjugated with anti-Rabbit-Cy3 (1:500, Jackson ImmunoResearch) for 2 hrs at room temperature. After washes with PBST, DAPI was added and sections were dried and cover-slipped. Confocal images were capitulated with Bio-Rad MRC 1024 confocal laser microscope. The use of Z-stack imaging was used to demonstrate co-localization. High resolution images were grayscaled and density was quantified using ImageJ 1.35s (NIH, USA). All procedures have been approved by IACUC committee of UC Irvine. AF487279 2 2 2 2 2 2

Chinese hamster ovary (CHO) cells stably expressing PKR2 photoprotein aequorin (PKR2-aq) were grown in alpha MEM Earl’s Salts (Invitrogen, Carlsbad, CA), containing 10% fetal bovine serum, 100 µg/ml streptomycin, 100 U/ml penicillin, G418 (100 µg/ml), and Zeocin (100 µg/ml). Human transient receptor cation channel, subtype V, member 2 (TRPV2) cDNA (ATCC) in pIRESpuro was transfected into PKR2-aq using lipofectAMINE manufactures protocol (Invitrogen, Carlsbad, CA). Clones of cells stably expressing PKR2 and TRPV2 were selected under 5µg/ml puromycin in a 5% COincubator at 37°C. Colonies were tested using the LB12 Sirius Luminometer (Berthold) as described []. 2 ¹⁹

Activation of TRPV2 has been shown to be induced by serum and thus all cellular experiments were pretreated with 6 hours serum-starvation []. Cells were serum starved at 37°C in B27 supplemented alpha MEM (Invitrogen, Carlsbad, CA) for 4 hours and in reduced serum OptiMEM I (Invitrogen, Carlsbad, CA) with coelenterazine cp (8µM) for an additional 2 hours. Stable cell lines were then trypsinized, counted, centrifuged, and resuspended in calcium-free HBSS (Hanks’Balanced Salt Solution) plus 10mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The luminescence of both PKR2 and PKR2-aq:TRPV2 stable cell lines was measured with a Berthold Luminometer as follows. ²⁰

After serum starvation, CHO cells were resuspended in Ca-free HBSS. Cell analysis occurred in a bioluminescence assay measuring changes in intracellular Ca([Ca]) concentrations both with application of HBSS or PK2. CHO cells were subjected to PK2 for 10 mins, followed by serum-free defined media. Cells were fixed at 0, 10, 30, 60, 90, and 120 mins and immunostained using extracellular-recognizing TRPV2 antibody. 2+ 2+ 2+ i

Linearized cDNA-containing vectors for PKR2 and TRPV2 were used to transcribe cRNA using mMessage mMachine reagents (Ambion, Austin, TX). Concentrations of cRNA were determined by spectrophotometric absorbance and by gel electrophoresis.

For oocyte isolation, sexually mature femalewere anaesthetized and lobes of ovaries were removed and stored at 16°C and gentamycin (Sigma). After oocytes were plucked and denuded by gentle vortexing with 1mg/ml collagenase (Worthington, Lakewood, NJ), healthy oocytes were injected with PKR2 cRNA or PKR2 and TRPV2 cRNAs. Xenopus laevis

oocytes were placed in a 35mm chamber with a bath solution containing 96mM NaCl and 2.5mM BaCl. The cells were impaled with two glass electrodes filled with 3M KCl connected to a two-electrode voltage clamp TEV-200 amplifier and oscilloscope. Once impaled, oocytes were incubated in various concentrations of Ruthenium Red for 5 minutes (Alexis, San Diego, CA) and were followed by PK2. For each experiment, 50nM PK2 was added for 40 seconds. All recordings and images were generated using pClamp 9 Software Suite (Axon, Union City, CA). Xenopus laveis 2

Data were analyzed using GraphPad Prism Software Version 5.0 (San Diego, CA). Two-way or one-way ANOVAs with Bonferroni post-hocs and unpaired t-test were used.

We screened the expression of all 33 known Trp channels in the mouse SCN [] byhybridization. The majority of the 33 known Trp channels were not expressed in the SCN, with several of them, including TRPC1, C4 and C5, being lightly expressed in the SCN (Supp. Fig.). The only robust expressed TRP channel was TRPV2 (Supp. Fig.). In addition to its robust expression in the SCN, TRPV2 was also expressed in the several known SCN target areas, such as paraventricular and dorsal medial nuclei of the hypothalamus. As the expression pattern of TRPV2 and PKR2 in the SCN and several known SCN targets is quite similar, we thus investigated whether PKR2 and TRPV2 are co-expressed in the same SCN neuron with combinedhybridization and immunostaining. Fig.shows that TRPV2 is indeed co-expressed with PKR2 in the SCN neurons. ^{21 22} S1 S1 1 in situ in situ

The circadian profile of TRPV2 expression in the SCN was then examined. Levels of TRPV2 mRNAs were observed to be non-oscillating in the SCN over a 24 hour period (Supp. Fig.). However, immunochemistry studies revealed a marked oscillation of TRPV2 protein expression in the SCN: TRPV2 protein level at CT4 was almost two fold higher than at CT16 (Fig.). As PKR2 is coexpressed with TRPV2 in the SCN neurons, we next examined whether the circadian oscillation of TRPV2 is linked to PK2 signaling with PK2-deficient (PK2-/-) mice. As shown in Fig., the oscillation of TRPV2 was absent in the SCN of PK2-/- mice: with the SCN at CT4 of PK2-/- mice displaying similarly low level as that of CT16 (Fig.). S2 2 2 2

To determine the functional interaction of PKR2 and TRPV2, we next examined Camobilization in stable cell lines that co-express PKR2 and TRPV2 (PKR2:TRPV2). As shown in Figure, the calcium mobilization signal was significantly increased in PKR2:TRPV2 cells, compared to parental PKR2 cells []. The modest mobilization of Cain the parental PKR2 CHO cell line can be attributed to calcium release from intracellular storage (Fig.and 19). Furthermore, ruthenium red, a TRPV blocker, was shown to dose-dependently inhibit the calcium signaling in PKR2:TRPV2 cells (Fig.). This decrease was due to blockade in TRPV2 channel activation, since [Ca]concentrations in the parental PKR2 cells were unaffected (Fig.). 2+ 2+ 2+ 3 ¹⁹ 3 3 3 i

We further used two-electrode voltage clamp electrophysiology to examine the functional interaction of TRPV2 and PKR2 inoocytes. In oocytes co-injected with PKR2 and TRPV2 cRNAs or injected with PKR2 alone, exposure to PK2 evoked inward currents (Fig.). Peak currents were about 5-fold higher in PKR2:TRPV2 injected oocytes compared with PKR2 alone (Fig., graph inset). Onset of currents occurred rapidly within ~5 secs in PKR2:TRPV2 oocytes with an aciculate profile. In contrast, with mean peak current amplitude of 400nA, PKR2-expressing oocytes had a slower PK2-induced onset of ~15 secs and displayed undulatory properties, typical of GPCR electrophysiological profiling []. Peaks returned to baseline more slowly in the PKR2-expressing oocytes and often displayed a prolonged oscillatory-IP3 patterning, also indicative of GPCR-driven signaling (Fig.). Xenopus 4 4 ²³ 4

To verify that the functionality of the TRPV2 channel is under the regulation of PKR2, we further used ruthenium red to block TRPV2 channel activity. In oocytes expressing PKR2:TRPV2, ruthenium red dose-dependently attenuated PK2-evoked currents (Fig., graph inset). In contrast, ruthenium red did not alter current amplitude in oocytes expressing PKR2 alone. These studies indicated that TRPV2 increases oocyte membrane current through PKR2 activation. 4

The molecular mechanism for the functional activation of TRPV2 is still unclear, with one likely possibility being that the TRPV2 channels are constitutively active and their activity is regulated by trafficking to cell surface expression []. We thus set out to examine the trafficking properties of TRPV2 in response to the PKR2 activation. In absence of the PKR2 activation, TRPV2 protein is low at the cell surface (Fig.). 30 mins after addition of PK2, TRPV2 was increased at the cellular membrane (Fig.). TRPV2 expression increases more by 60 min, and peaks at about 90 min (Fig.). Interestingly, robust expression levels of TRPV2 were observed in the formation of large clusters on the membrane, particularly around peak level (Fig.). Confocal images of Z-stack (Supp. Fig.) clearly demonstrated the robust trafficking of TRPV2 to the cell surface membrane via the activation of PKR2. To further ascertain this trafficking to cell surface expression of TRPV2 was truly due to the activation of PKR2, we pretreated the cells with a selective PKR2 antagonist, PKRA7 []. Fig.shows that PKRA7 blocked the cell surface expression of TRPV2 induced by PKR2 activation. Taken together, these results indicate that PKR2 activation increases the trafficking of TRPV2 to cell surface. ^{20 24 25 26 27 28 29} 5 5 5 5 S3 ^{30 31} 5