Alternative Splicing of the Pituitary Adenylate Cyclase-Activating Polypeptide Receptor PAC1: Mechanisms of Fine Tuning of Brain Activity

Alternative splicing of the precursor mRNA encoding for the neuropeptide receptor PAC1/ADCYAP1R1 generates multiple protein products that exhibit pleiotropic activities. Recent studies in mammals and zebrafish have implicated some of these splice isoforms in control of both cellular and body homeostasis. Here, we review the regulation of PAC1 splice variants and their underlying signal transduction and physiological processes in the nervous system.


INTRODUCTION
G-protein-coupled receptors (GPCRs) represent the largest family of membrane-associated proteins mediating physiological responses in vertebrates by means of controlling metabolic, neural, and developmental functions (Lefkowitz, 2007;Frooninckx et al., 2012;Zhang and Eggert, 2013). These proteins are expressed in almost all types of tissues and are represented by over 1,000 membrane receptors for extracellular (EC) ligands including hormones, neurotransmitters, pheromones, lipids, and other proteins (Xue et al., 2008;Markovic and Grammatopoulos, 2009;Nordstrom et al., 2009;Hoyer and Bartfai, 2012;Jafurulla and Chattopadhyay, 2013). The basic structure of GPCR proteins consists of a seven-transmembrane (TM) domain with a complex EC structure composed of an N-terminal region and three EC loops involved in the diverse ligand recognition process. Three intracellular (IC) loops and a C-terminal domain transduce a signal into the cell's cytoplasm and nucleus. Typically, ligand binding to the EC loops induces conformational changes in the TM and IC domains of the receptor resulting in specific coupling to a set of cytoplasmic molecules, termed G-proteins, each composed of different isoforms of alpha, beta, and gamma subunits. G-proteins in turn regulate the activity of IC effector molecules, including adenylate cyclase (AC), phospholipase Cβ (PLC), and RhoGEF causing the activation of secondary messengers such as cyclic AMP (cAMP), inositol-1,4,5triphosphate (InP3), and diacylglycerol leading to the initiation of distinct IC signaling pathways (Simon et al., 1991;Sah et al., 2000;Pierce et al., 2002;Oldham and Hamm, 2008;Mizuno and Itoh, 2009;Mahata et al., 2011;Maurice et al., 2011).
The diversity of the GPCR-initiated signal transduction pathways is determined by the multiplicity of cognate ligands with different receptor binding properties and by the receptors' membrane-interacting partners that form homo-and hetero-dimerized GPCR complexes. These receptor dimers have unique binding properties to both EC ligands and a set of IC G-proteins (Milligan and Kostenis, 2006;Harikumar et al., 2008;Furness et al., 2012). Thus, ligand binding, along with receptor heterodimerization, can generate diversity in G-proteins coupling that can generate varied IC signaling functions.
Beyond the complexity of the aforementioned EC and IC GPCRs interacting effectors, the genetic diversity of the GPCR family can be generated by means of alternative precursor mRNAs (pre-mRNA) splicing of exons encoding specific protein moieties and causing considerable functional differences of the resulting splicing products (Black, 2003;Jaillon et al., 2008;Furness et al., 2012). Alternative splicing mechanisms allow the generation of multiple mRNA transcript variants from a single gene by utilizing different combinations of exons by means of skipping or insertion of alternatively spliced exons. In particular, neuronal cells are known to exhibit high levels of alternative splicing, generating the basis for molecular and cellular diversity important for the patterning and maintenance of the central and peripheral nervous systems (Yeo et al., 2007;Betke et al., 2012;Norris and Calarco, 2012;Sun et al., 2012). In the case of GPCRs, this combinatorial exon assembly can lead to changes in the protein domains responsible for ligand binding, IC effector coupling, as well as receptor stability, and endocytosis.
In this review we focus on one GPCR, the pituitary ACactivating polypeptide (PACAP) receptor (ADCAYP1R1), also known as PAC1. This receptor represents a fascinating example of how alternative splicing of a single GPCR gene leads to different physiological outcomes. We will describe the current knowledge regarding the role and mechanism of action of PAC1 splice variants in development, physiology, and diseases focusing on PAC1's role in the nervous system.

THE PAC1 RECEPTOR
PAC1 belongs to the glucagon/secretin receptor family of GPCRs that consists of hormone and neuropeptide receptors. These receptors signal through coupling to the G-protein alpha subunits Gs and Gq that typically activate AC and phospholipase Cβ enzymes, respectively (McCulloch et al., 2002;Ahren, 2008;Dickson and Finlayson, 2009;Vallejo, 2009;Vaudry et al., 2009). Coupling of PAC1 to Gs may also lead to cAMPdependent accumulation of IC calcium (Braas and May, 1996;Mustafa et al., 2010). The PAC1 gene contains multiple exons that undergo extensive alternative splicing. The peptide PACAP is the high-affinity ligand for PAC1. Post-translational proteolytic processing of the PACAP precursor protein generates several polypeptides with varying sizes, including PACAP38 and PACAP27 (Miyata et al., 1989;Vaudry et al., 2009;Harmar et al., 2012;Watkins et al., 2012). PAC1 was found to play a pivotal role in the spatio-temporal regulation of proliferation, differentiation, or cell survival during development as well as in the regulation of synthesis and release of neuroendocrine hormones.
Phylogenetic analysis of the vertebrate PAC1 receptor family indicates their origin from a common ancestral gene and demonstrates a tree topology with species-specific paralogs belonging to different separated sub-groups (Figure 1). Bony fishes (teleosts) genomes typically contain more than one gene due to teleostspecific gene duplication event of both ligand and receptor molecules during the evolution of these species (Wei et al., 1998;Fradinger et al., 2005;Bjarnadottir et al., 2006;Cardoso et al., 2007;Machluf et al., 2011).

ALTERNATIVELY SPLICED PAC1 GENE PRODUCTS
The alternative splicing of PAC1 has been extensively studied. In this review we use the exon numbers designation of the human PAC1 and otherwise indicate cases where the exons numbers do not match. The human gene was shown to contain 18 exons with the open reading frame encoded by exons 2-18 (Chatterjee et al., 1997;Lutz et al., 2006). Ten of these exons are constitutively expressed (exons 2, 3, 7-13, 18), whereas the rest (exons 4-6, 14, 15, and possibly 16, 17) are regulated by the alternative splicing (Figure 2). The N-terminal part of PAC1's EC domain is encoded by six exons. Exons 7-17 encode the seven-TM domains including EC and IC loops and exon 18 encodes the C-terminal cytoplasmic tail including the 3 -untranslated region (Figures 2  and 3). PAC1 splicing variants were identified in other vertebrate species including rat, mouse, frog, and fish. In Table 1 we assembled all known PAC1 splice isoforms from different vertebrate species. The functional outcome of these splicing events will be discussed later. By and large, PAC1 alternative splicing can be divided into four types of splicing events that impact receptor functions (Figure 3; Table 1): (1) Variations in the EC N-terminal domain altering the ligand-binding specificity and affinity.
(2) Variations in exons encoding to part of the third IC loop (IC3) thereby affecting G-protein coupling and/or interaction with other IC signaling proteins. (3) Variations in the TM domains TM2 and TM4 contributing to the receptors heteromerization and IC transport. (4) Variations in the 5 UTR that may affect mRNA expression dynamics. Notably, splicing products containing different combinations of N-terminal and IC3 splice variants were identified in mammalian species.
The terminology used in independent studies can be somewhat perplexing as different names were designated to PAC1 isoforms corresponding to the same splicing events identified in various species. In the following section we describe the various splice variants and indicate when a different (i.e., specie-specific) name was designated to the same isoform. A splice isoform that includes all N-terminal encoding exons but does not contain IC (IC3) insertion(s) is referred to as "PAC1-null" ( Table 1). The ligand binding and signaling properties of PAC1-null are often used as a reference when assessing the activities of other splice variants.

INTRACELLULAR LOOP VARIATIONS
PAC1 splice variants in the third IC loop (IC3) have been identified in human, amphibian, fishes, and rodents. These splice isoforms are characterized by the presence of one or two cassettes of 84 nucleotides (hip or hop1 variants) or 81 nucleotides (hop2 variant), or a combination of alternative spliced "cassettes" (hip-hop1 or hip-hop2) (Spengler et al., 1993;Journot et al., 1995;Grimaldi and Cavallaro, 1999;McCulloch et al., 2001;Ronaldson et al., 2002;Fradinger et al., 2005;Ushiyama et al., 2007;May et al., 2010;Holighaus et al., 2011). In human, hip and hop variants are also referred to as SV1 and SV2 or SV3 for hip-hop1 . In addition, a splice variant formed by a C-terminal deletion of 193 nucleotides (denoted"hop1 novel") including two amino acids that are essential for G-protein recognition was identified in rat cochlea samples (Abu-Hamdan et al., 2006). Similar alternative splicing variants of PAC1 are also found in non-mammalian vertebrates such as the frog and bony fishes. Three alternative splice variants were identified in the frog IC3 loop (Alexandre et al., 2002). PAC1-R25, with an insertion of 25 amino acids, corresponds to the mammalian hop cassette. PAC1-R41 contains a cassette with no homology to any other variant. PAC1-RMc with a unique cytoplasmic insertion of 13 amino acid into the TM7 domain is missing a canonical Gs recognition motif. PAC1 genes were also identified in zebrafish, goldfish, stickleback, fugu, sea bream, and several others bony fish species. Three zebrafish PAC1 alternative splice variants with insertions in the IC3 loop were identified. Two of them were found to be homologous to the hop1 (84 nucleotides) and hop2 (81 nucleotides) mammalian isoforms (Fradinger et al., 2005). A unique 107 nucleotides inclusion with a premature stop codon (PAC1-skip) resulting in a truncated PAC1 protein did not correspond to any previously detected PAC1 isoform (Fradinger et al., 2005). This isoform resembles an alternative splicing event in a human gene encoding another secretin family receptor, VPAC1. Interestingly, the hip cassette variant was not identified in the mouse and zebrafish genomes. Moreover, teleosts genomes contain two (duplicated) PAC1 paralogs, pac1a and pac1b, however, only pac1a encodes for the hop splicing cassette.

TRANSMEMBRANAL DOMAIN VARIATIONS
A PAC1 variant, cloned from the rat cerebellum, has amino acids deletion/substitution in the TM4 domain along with two amino acid substitutions in the N-terminal (D136N) and TM2 (N190D) domains (Chatterjee et al., 1996;Ajpru et al., 2002). The exact molecular mechanism underlying these variations remains unclear. Notably, the TM4 domain of the secretin family receptors are involved in homo-and hetero-oligomerization of these receptors, associations with receptor activity-modifying proteins (RAMPs), and with GPCR kinases (GRKs) thereby suggesting that splice alterations in the PAC1-TM4 domain may affect receptor function (Morfis et al., 2003;Ritter and Hall, 2009;Magalhaes et al., 2012).

UTR VARIATIONS
Alternative splicing events in exons located at the 5 UTR were identified for rat PAC1 gene (Chatterjee et al., 1997). These include different alternative usage of exons located upstream to the ATG translation start codon. Such variations in the 5 UTR organization and sequences may play a role in the regulation of mRNA expression.

ALTERNATIVE PAC1 SPLICING ALTERS LIGAND-BINDING PROPERTIES
PAC1 is considered as being the high-affinity receptor for PACAP, while it displays low binding affinity to the vasoactive intestinal polypeptide (VIP) (Apostolakis et al., 2005;Vaudry et al., 2009;Harmar et al., 2012). Alternative splicing of PAC1 results in different protein products displaying different ligand-binding properties that may result in changes in affinity and selectivity ( Table 1). Most studies use the PAC1-null (McCulloch et al., 2001;Holighaus et al., 2011) isoform as a reference for PACAP and VIP binding properties.
Notably, binding analysis of alternative spliced isoforms, which result from combined changes in the EC and IC protein domains yields interesting results. Thus, a splice variant composed of PAC1short with the inclusion of hop1-cassette, displays similar PACAP and VIP binding properties to that of PAC1-null and different VIP binding properties than PAC1-short alone (Lutz et al., 2006;Ushiyama et al., 2007Ushiyama et al., , 2010. Another combined splice variant composed of PAC1-δ5 or PAC1-δ5,6 together with the hip cassette had different ligand-binding properties to that of PAC1, PAC1-δ5, or PAC1-hip alone (Lutz et al., 2006) (Table 1). This imply that the IC3 domain contributes to the association of PAC1 with its cognate ligands.
These results may be explained by a model for agonist binding to the corticotropin-releasing hormone (CRH) receptor, which belongs to the same GPCR sub-family of PAC1 (Dong et al., 2005). According to this model the TM6 protein domain plays a structural role in ligand binding. Thus, insertions into IC3 loop, which is located between TM5 and TM6 region may cause a conformational change in TM6 and possibility affect binding of PACAP and VIP to the EC part of the receptor. A proof for this model awaits a solved three-dimensional protein structural of the different PAC1 isoforms.
Couvineau and Laburthe, 2012) (Figures 3 and 4). In addition, PAC1 was shown to regulate the level of IC Ca 2+ in either Gprotein dependent or independent manners. The following section reviews the role of PAC1 splice variants in the activation of the above pathways as well as other non-canonical signaling cascades (see also Table 1).
Consistent with the effect of IC3 loop insertions on ligandbinding properties, hip-and hop2-cassettes conferred comparable potency to induction of cAMP and IP3 accumulation by PACAP and VIP but at the same time characterized by much lower efficacy of this response (Spengler et al., 1993;Ronaldson et al., 2002;Lutz et al., 2006). On the other hand, PAC1-hip variant was shown to retain AC mediated signaling but demonstrated impaired coupling to the PLCβ pathway (May et al., 2010).
PAC1-null was reported to modulate cytosolic Ca 2+ mobilization from both EC and IC stores (Shioda et al., 1997;Masmoudi et al., 2003;Payet et al., 2003;Nishimoto et al., 2007). In acutely dissociated rat melanotrophs, the increase of cytosolic Ca 2+ was dependent on the activation of non-selective cation channels and the facilitation of voltage-dependent Ca 2+ channels by PKCand PKA-dependent phosphorylation, respectively (Tanaka et al., 1997). cAMP-dependent entry of EC calcium was also reported in astrocyte cells (Vallejo, 2009). It was also suggested that PAC1null-mediated elevation of cytoplasmic Ca 2+ levels in NG108-15 cells is due to IP 3 receptor-mediated Ca 2+ release from IC stores (Holighaus et al., 2011).
The PAC1-hop1 variant is responsible for both Ca 2+ mobilization from IC stores and influx through voltage-gated Ca 2+ channels in bovine chromaffin cells (Mustafa et al., 2007). Transfection of PAC1-hop to the adrenomedullary pheochromocytoma (PC12) cell line showed sustained IP 3 -mediated Ca 2+ release from IC stores and from store-operated Ca 2+ entry (SOCE) (Taupenot et al., 1999;Mustafa et al., 2007Mustafa et al., , 2010. In bovine adrenal chromaffin cells, PAC1-hop was shown to mediate cytosolic Ca 2+ release from ryanodine/caffeine-sensitive Ca 2+ stores that was not dependent on either cAMP or IP3 generation (Tanaka et al., 1998;Payet et al., 2003). Heterologous expression of the rat PAC1-hop1 but not PAC1-hip variant in NG108-15 and PC12 cells leads to an increase in IC Ca 2+ concentration (Mustafa et al., 2007;Holighaus et al., 2011). In both cell lines the response consisted of a rapid and transient rise of Ca 2+ reminiscent of IP 3 receptor-mediated Ca 2+ release from IC stores followed by a prolonged Ca 2+ accumulated from EC source.
When compared with PAC1-null, PAC1-hop1 was more potent in Ca 2+ elevation (Holighaus et al., 2011). However, following overexpression in Chinese Hamster Ovarian cells, activation of PAC1-null induced higher Ca 2+ levels when compared with activation of PAC1-hop1 (Ushiyama et al., 2010). PAC1-hop1 also mediated PACAP-induced Ca 2+ release from ER and PKCγ translocation to the nucleus and plasma membrane resulting in the astrocytic differentiation (Nicot and DiCicco-Bloom, 2001). Finally, stimulation of voltage-gated L-type or non L-type Ca 2+ channels following initiation of PAC1-hop1 signaling was also reported (Mustafa et al., 2010).
Somewhat limited data was reported with regards to Ca 2+ signaling via other PAC1 splice variants. Thus, δ5,6/short as well as the PAC1 isoform containing combined "short" deletion in the N-terminal EC1 domain along with hop1-cassette in the IC3 loop exhibit PACAP-induced cytoplasmic calcium elevation (Ushiyama et al., 2010). PAC1-3a was shown to induce Ca 2+ accumulation through coupling to Gs/cAMP rather than Gq/PLCβ pathway (Pilzer and Gozes, 2006a). PAC1-TM4 that was found to be inactive when assayed for both cAMP and PLCβ activation but it displayed elevation of Ca 2+ in response to PACAP27. It was demonstrated www.frontiersin.org that this effect involved the modulation of voltage-gated L-type Ca 2+ channels (Chatterjee et al., 1996).
Taken together, the apparent inconsistent results concerning the mechanisms underlying the aforementioned Ca 2+ signaling events are most likely due to the different cellular systems (e.g., cell types) employed by the above studies to analyze PAC1-mediated IC Ca 2+ changes.

EXPRESSION OF PAC1 SPLICE ISOFORMS IN THE NERVOUS SYSTEM
Information on time-and region-specific distribution of PAC1 splice isoforms may shed light on how PAC1 gene products regulate a plethora of biological functions in developmental and physiological processes (D'Agata et al., 1996;Waschek et al., 2000;Waschek, 2003;Vaudry et al., 2009). The distribution of PAC1 has been examined in many species using different techniques that revealed widespread expression in different tissues, including the nervous, cardiovascular, endocrine, immune, and respiratory systems (Aino et al., 1995;Abu-Hamdan et al., 2006;Gomariz et al., 2006;Molnar et al., 2008;Jolivel et al., 2009;Shneider et al., 2010;Lugo et al., 2011;Buljan et al., 2012). However, knowledge concerning the expression of PAC1 splice variants is somewhat limited at it is mainly based on analyses of isolated brain areas and/or primary cell cultures. The reported changes in PAC1 receptor variant expression appear to be most evident during development. We have summarized the existing data concerning the expression of these variants by focusing on expression in the nervous system of different mammalian species.
The major mRNA isoform of PAC1 in the brain is PAC1-null, which contains no splice deletions or insertions. PAC1-null is predominantly expressed in neurons residing in different brain areas although it is also detected in glial cells of the cerebella cortex and in activated astrocytes (Pilzer and Gozes, 2006b;Dickson and Finlayson, 2009;Vaudry et al., 2009). PAC1 variants with N-terminal deletions are well represented in human fetal brain tissues and in five human neuroblastoma lines (Lutz et al., 2006;Falktoft et al., 2009), thereby suggesting their role in immature nervous tissue. N-terminal splice variants were detected in different brain areas. The PAC1-short variant with 21 amino acids N-terminal deletion was predominantly found in the thalamus, hypothalamus, and the hypophysis, and more moderately in the amygdala and retina (Dautzenberg et al., 1999;Grimaldi and Cavallaro, 1999;Jamen et al., 2002;Girard et al., 2004;Lutz et al., 2006;Ushiyama et al., 2007Ushiyama et al., , 2010Hammack et al., 2010a). It was shown to be expressed in cochlea subfractions along with the PAC1-δ5 isoform with a seven amino acid deletion (Abu-Hamdan et al., 2006). The PAC1-very short variant (57 amino acid deletion) was also detected at low levels in the mouse amygdala and cortex though not in the retina. All three N-terminal variants (null, short, and very short) were abundantly expressed in neuronal tissues (Dickson and Finlayson, 2009;Vaudry et al., 2009). The PAC1-3a variant containing N-terminal insertion of 24 amino acids was detected in rat cerebral cortex. This splice isoform is also highly expressed in distinct cell populations of the testis, including Sertoli cells, pachytene spermatocytes, and round spermatids (Daniel et al., 2001;Ajpru et al., 2002;Pilzer and Gozes, 2006a).
Contrary to the aforementioned N-terminal splice variants that were only reported in mammals, IC loops (i.e., IC3) splice products are found in all studied vertebrate species. In the postnatal cerebral cortex of rats, the expression of PAC1-null, hop1, hip, and hip-hop1 dramatically decreases during the first neonatal month suggesting a major role for these isoforms in embryonic development (Shneider et al., 2010). Interestingly, the expression of these isoforms was shown to be gender dependent displaying higher levels in female postnatal brain at least during the first 3 months of development. In the developing retina, the proportion of IC3 PAC1 isoforms changes as development proceeds: PAC1-hip-hop1 transcript demonstrated transient elevation at day P10, while a decrease in PAC1-null and hip along with elevation of PAC1-hop1 level was observed by P20 (Lakk et al., 2012). PAC1-hop1 expression was found in the olfactory bulb, hippocampus, cerebral cortex, cerebellum, and striatum of newborn rats. PAC1-hop2 was present in rat cerebral astrocytes from newborn brains, in neuronal enriched cultures, and in PC12 cells that undergo neuronal differentiation following NGF treatment (Jamen et al., 2002;Onoue et al., 2002;Hashimoto et al., 2003;Ravni et al., 2006;Mustafa et al., 2010). PAC1-hip was detected at much lower levels in adult tissues and it was therefore speculated to be important for the embryonic development (Shneider et al., 2010;Holighaus et al., 2011). The relative expression of human and rat PAC1 splice variants in the frontal cortex was reported to have the following expression level hierarchy: null > SV1/hip > SV2/hop = SV3/hiphop Germano et al., 2004;Lutz et al., 2006). PAC1-hop1 was found to be expressed in all neuroendocrine cells, suggesting a fine tuning of PAC1-mediated signaling in the neuroendocrine cells (Mustafa et al., 2010). PAC1-hop1 splice isoform was the major form expressed in the superior cervical ganglia (SCG) sympathetic neurons, which also express PAC1-short (Braas and May, 1999). Zebrafish IC3 splice isoforms are widely expressed in the adult tissues with PAC1-hop1 detected in brain and testis, PAC1-hop2 in the ovary and PAC1-skip variant in the gills (Fradinger et al., 2005). Lastly, although PAC1-TM4 isoform is mainly found in pancreatic β-cells it is also expressed in the cerebral cortex, cerebellum, and brain stem (Chatterjee et al., 1996;Ajpru et al., 2002).
In summary, changes in PAC1 receptor variant expression appear to be most evident during development. The fact that PAC1 splice variants display differential expression patterns in the nervous system suggests that alternative splicing of this gene product plays a role in fine tuning of PAC1 activity in these areas.

REGULATION OF PAC1 SPLICING BY NEURONAL-SPECIFIC RNA-BINDING PROTEINS
The regulation of PAC1 splicing is not well understood. Alternative splicing of pre-mRNA is often regulated by conserved cis-regulatory RNA sequence elements that serve as recognition sites for different splicing factors. These RNA-binding splicing factors differ from the general spliceosome machinery proteins due to the formers' ability to either activate or repress the inclusion of alternatively spliced exons, depending on whether the factors' binding site is located upstream, downstream, or inside the exon. Neuronal-specific splicing factors include nova-1/2, Rbfox-1/2, and ELKV2 splicing factors, the neuronal-specific polypyrimidine tract-binding protein (nPTB) as well as a set of heterogeneous nuclear ribonucleoproteins (hnRNP A1,L,F.H1). As the consensus cis-acting binding sequences for the aforementioned splicing factors have been identified, their involvement in regulating the alternative splicing of a given gene by sequence analysis can be predicted. We analyzed the presence of known consensus binding sites for the following RNA-binding proteins: Rbfox-1/2, Nova-1/2 (single and repeatedly organized elements), SC-35, nPTB enhancers and silencers, and CaRRE for Ca 2+ /CaM kinase IV recognition sequences. Putative binding sites for neuronal-specific splicing factors in the respective PAC1 genes of human, mouse, and zebrafish are shown in Figure 5.

FIGURE 5 | Regulation of PAC1 splicing. (A)
A chart depicting the predicted binding sites for neuronal-specific RNA-binding proteins that potentially regulate PAC1 gene splicing by binding in proximity to PAC1 exons. Consensus cis-acting binding DNA elements for the respective RNA-binding protein are depicted with respect to their position in the PAC1 genes of rat, mouse, human, and zebrafish PAC1. We analyzed the presence of putative consensus binding sites located within 300 base-pairs upstream (upst) or downstream (dnst) and inside the exons. (B) Schematic representation of the PAC1 gene in which the location of the analyzed cis-acting elements is color-coded as above. Corresponding exons numbers and sizes (bp) are indicated.

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Rbfox-1/2 can act either as a splicing enhancer or as an inducer of exon skipping (Zhang et al., 2008). Putative Rbfox-1/2 binding sites located within a short distance downstream of target exons are predicted to induce exon inclusion whereas upstream binding sites are predicted to cause exon skipping. Such Rbfox-1/2 recognition element located within a short distance downstream of hop1 encoding exon has been experimentally validated as a genuine Rbfox-1 binding site (Lee et al., 2009). Our own PAC1 bioinformatic analysis identified a putative Rbfox-1/2 binding element within −30 nucleotides downstream from conserved exon spanning zebrafish TM7 suggesting that this domain might be regulated by alternative splicing (Figure 5). Rbfox-1/2 motifs are also present in the sequences of all examined species downstream of exon 16 encoding parts of the TM7 domain. Another recognition site is located upstream of exon 4 consistent with a deletion of the exon resulting in the generation of PAC1-very short splice variant (Figure 5). Rbfox-1/2 recognition motif predicts potential deletion of exon 8 that may result in a generation of a soluble PAC1 receptor as in the case of the CRH receptor Slominski, 2009, 2010). Rbfox-1/2 binding site is also detected downstream of exon 11, which is known to encode the most hypervariable amino acid sequence among Secretin family GPCRs, as well as part of TM4 domain which undergoes alternative splicing in PAC1-TM4 isoform (Markovic and Grammatopoulos, 2009).
Calcium signaling plays an important role in neuronal development and is involved in the regulation of immediate and long-term neuronal responses to various stimuli such as stressors and hormones (Ghosh et al., 1994;West et al., 2001). Depolarization is known to be critical for modulating the neuronal activity that induces Ca 2+ -dependent gene regulation, including alternative splicing. Ca 2+ -dependent splicing is mediated by L-type calcium channels and by Ca 2+ /calmodulin-dependent protein kinases IV (CaMK IV) (Lee et al., 2007(Lee et al., , 2009. The latter was shown to repress splicing of target genes containing specific recognition elements (CaRRE) located within the 3 splice site or inside the exon. Our own analysis detected additional putative CaRREs that can be responsive to CaMK IV. We predicted single, multiple, and tandemly organized CaRRE repeats upstream of exon 15 and 17 of the zebrafish, human, and mouse PAC1 receptors, suggesting the involvement of CaMK IV in regulating the alternative splicing of these exons. Putative CaRREs are also present proximal to mouse, rat, and zebrafish exon 4 ( Figure 5).
Exonic and intronic hnRNPA1 and nPTB cis-elements that are important for activity-dependent splicing in neuronal cells were shown to repress the inclusion of target exons (Allemand et al., 2005;An and Grabowski, 2007;Donev et al., 2007;Resnick et al., 2008;Zheng et al., 2012). Putative hnRNPA1 and nPTB recognition sites are located proximal to exons 4, 5, and 6 consistent with the known deletions of these exons that result in the generation of PAC1-short and very short splice variants. Putative hnRNPA1 recognition motifs are also found near exons 8-10 encoding to IC1, IC2, and EC2 domains (Figure 5).
Nova-1/2 regulates target exon inclusion or skipping with broad distribution of binding sites across the gene sequence (Ule et al., 2005;Yano et al., 2010;Norris and Calarco, 2012). Predicted Nova-1/2 recognition elements are found in human and mouse PAC1 and in both paralogs of zebrafish PAC1 gene (1a and 1b). Interestingly, higher frequency of putative splicing-inducing AUrich and Nova-specific sequences are found in intronic regions upstream of alternative spliced exons characteristic of the Secretin family splicing patterns, e.g., around exon 4, 8, 9, 10 (Zmijewski and Slominski, 2009) as well as exon 17. Exons 4-6 encode sequences covering known N-terminal PAC1 deletion regions (deletions of 7, 21, and 57 amino acids). Exon 17 encodes the last amino acids of the seventh TM domain and the beginning of the IC tail shown to represent the highly conserved exon within the secretin family (Markovic and Grammatopoulos, 2009). Skipping of this exon was not detected in the PAC1 receptor gene, but it has been proposed that such exon skipping in the human CRF1 and PTH1 receptors results in a six TM receptor displaying impaired trafficking (Shyu et al., 1996;Grammatopoulos et al., 1999;Markovic et al., 2008). The rabbit calcitonin receptor is also formed as a result of the skipping of the TM7 domain and displays slightly diminished cAMP activation along with deficiency in IP3 induction (Shyu et al., 1996). Taken together the above analysis of potential cis-elements spanning TM7 exons predicts that a PAC1 splice variant containing TM7 skipping may exist in neuronal tissues.
An important question raised in this chapter is whether the aforementioned RNA-binding factors regulate PAC1 splicing in vivo. It has been demonstrated that alternative splicing of the PAC1 receptor is regulated by the Rbfox-1 splicing factor in depolarized neurons. In this case, neuronal depolarization induced CaM-kinase dependent self-splicing of Rbfox-1, leading to the translocation of Rbfox-1/2 from the cytoplasm to the nucleus where Rbfox-1 could mediate the alternative splicing of neuronal-specific target genes, including PAC1. Interestingly, Rbfox-1 mRNA levels were shown to be regulated by the Orthopedia (Otp) homedomain-containing protein. The latter is a hypothalamic-specific transcriptional factor, which plays a role in hypothalamic neuronal-specification during development and in regulation of body homeostasis in the mature zebrafish brain (Acampora et al., 1999;Wang and Lufkin, 2000;Blechman et al., 2007;Ryu et al., 2007;Amir-Zilberstein et al., 2012;Fernandes et al., 2012). The involvement and regulation of other neuronal-specific RNA-binding factors in PAC1 splicing remains to be determined.

PLEIOTROPIC ROLES OF PAC1 SPLICE ISOFORMS IN THE NERVOUS SYSTEM
The distribution of PAC1 and its ligands PACAP and VIP in a variety of cell types is reminiscent of the pleiotropic functions of PAC1 in development and physiology. PACAP is described in the literature as a hormone, neuropeptide, endocrine peptide, neurotransmitter, and neurotrophic factor. It affects the central nervous system (CNS), cardiovascular system, pituitary, thyroid, and adrenal glands and provides functional activities in the gonads, gastrointestinal tract, and pancreas (Waschek et al., 2000;Dickson and Finlayson, 2009;Vaudry et al., 2009). In the nervous system, PAC1 was shown to affect a variety of hormones and neuropeptides, including stimulation of oxytocin (Jamen et al., 2003) and melatonin (Nakahara et al., 2002) release and de novo mRNA synthesis of CRH (Nakahara et al., 2002;Amir-Zilberstein et al., 2012), arginine-vasopressin (Murase et al., 1995;Gillard et al., 2006), GnRH (Kanasaki et al., 2009;Winters and Moore, 2011), somatostatin (Kageyama et al., 2007), and MSH (Mounien et al., 2006). This overabundance of PAC1-mediated physiological activities can be made possible through the signaling diversity of its alternatively spliced gene products. Moreover, different expression of PAC1 isoforms is common in neuronal ontogeny. Such differences in the expression of PAC1 splice variants might modulate final outputs of VIP and PACAP activities as neurotransmitters, neurotrophic, or differentiation factors. Examples for the involvement of PAC1 splice isoforms in mediating these activities are described below.

NEUROGENESIS, NEUROPROTECTION, AND DIFFERENTIATION
Neural progenitor cells (NPC) and astroglial cells express PAC1null and PAC1-hop1 variants that mediate both cAMP-and Ca 2+dependent signaling pathways and induce production of a subset of neurotrophic factors resulting in neuronal proliferation and/or differentiation. PAC1-hop2 variant, detected in astrogenic and neuronal populations, promote neuroprotective function induced by VIP. In another study, VIP was also demonstrated to induce PAC1-hop2-mediated astrocytes neuroprotection against oxidative stress. Astrocytic expression of PAC1-hop2 isoform may therefore play a critical role in the NPC shift toward neuronal or astrocytes differentiation (Ashur-Fabian et al., 1997;Pilzer and Gozes, 2006b).
Expression of PAC1-null and PAC1-hop1 define region-specific neurogenesis in the CNS and peripheral nervous systems (Lu et al., 1998). Both variants are differently expressed in proliferating sympathetic (PAC1-hop1) and cortical precursors (PAC1-null) revealing opposing PACAP-mediated mitogenic regulation -either by stimulating sympathetic neuroblast proliferation or by inhibiting cortical precursor mitosis. Ectopic expression of PAC1-hop1 in cortical neuroblasts may transform the anti-mitotic effect of PACAP into promitotic. This promitotic signaling was shown to involve PLC signaling pathway (Nicot and DiCicco-Bloom, 2001). PAC1-hop1 was also shown to promote sympathetic neurons survival following growth factor withdrawal (May et al., 2010).
In conjunction with the abovementioned, PACAP may possess therapeutic potential for neurodegenerative pathologies, such as Parkinson's and Alzheimer's disease (AD). PACAP is enriched in rat mesencephalic dopaminergic neurons and protects them from neurotoxin-induced death (Reglodi et al., 2004). PACAP was shown to be downregulated in several mouse models for AD as well as in the human temporal cortex of AD patients (Kojro et al., 2006;Rat et al., 2011;Postina, 2012). Amyloidogenic processing of the amyloid precursor protein (APP) to αβ-peptides is responsible for the development of AD. However, non-amyloidogenic APP processing pathway results in the α-secretase-dependent cleavage within the αβ-peptide region, preventing AD pathology. Continual PACAP and PAC1 activation resulted also in a feed-forward autocrine elevation of both PACAP and PAC1 in mice that may further facilitate non-amyloidogenic APP cleavage. α-secretase activation was shown to be regulated by ERK1 and ERK 2 and PI-3 kinase, suggesting that PAC1-hop1 is the most efficient isoform with regard to the activation of these downstream PLC effectors (Kojro et al., 2006;Rat et al., 2011;Postina, 2012). It remains to be determined whether specific PAC1 splice variants are involved in these neuroprotective activities.

NEUROSECRETION AND NEUROTRANSMISSION
Pituitary AC-activating polypeptide was shown to elicit catecholamine synthesis and release. Expression of PAC1-hop1 in bovine chromaffin NG108-15 cell line, which lacks endogenous PAC1 receptors, induces the release of norepinephrine via a Ca 2+ influx-dependent mechanism (Mustafa et al., 2007). Transfected PAC1-hop1 triggers sustained catecholamine secretion by regulating Ca 2+ levels through both ER and EC Ca 2+ channels (Mustafa et al., 2010;Smith and Eiden, 2012). Moreover, acute and longterm met-enkephalin secretion and enkephalin biosynthesis were attributed to bovine chromaffin cells upon PAC1-hop1-mediated activation of L-type Ca 2+ channels. This implies a role of hop1 splice cassette in regulating neuroendocrine secretion. SCG sympathetic neurons were shown to predominantly express the PAC1-hop1 splice isoform and PACAP stimulates neuropeptide Y release in these neurons via a mechanism involving both AC and PLCβ (Braas and May, 1999).
Pituitary AC-activating polypeptide/PAC1 signaling was recently implicated in abnormal stress responses underlying posttraumatic stress disorder (PTSD) pathology (Ressler et al., 2011;Andero and Ressler, 2012;Hauger et al., 2012). PACAP38 levels in females were strongly associated with PTSD symptoms. Ressler et al. and May et al. (2010) found that a single nucleotide polymorphism (SNP), rs2267735 in the PAC1 gene is correlated with PTSD in a gender-specific estrogen-dependent manner. This SNP, which resides in the putative estrogen response element within PAC1's promoter/enhancer is associated with sexdependent PTSD, fear discrimination, PAC1 mRNA expression, and methylation of PAC1. Using mouse models, these authors showed that PAC1 mRNA was induced by fear conditioning or estrogen hormone replacement in the bed nucleus of stria terminalis (BNST), which is a component of the extended amygdala involved in fear-and anxiety-like responses.
In connection to the above, steroid-induced changes in PAC1 splice isoforms were demonstrated in the case of PAC1-hop2 (Apostolakis et al., 2005). PAC1-hop2 and the N-terminal PAC1short splice variants were detected in the hypothalamic ventromedial nucleus (VMN) upon estradiol or dual estradiol/progesterone www.frontiersin.org treatment. The ratio of PAC1-hop2 mRNA to other PAC1 variants in VMN depends on steroid application, implying its importance for cumulative PAC1 isoforms expression and hence signaling properties. PACAP-induced sexual/mating behavior in female rats was dependent on progesterone receptor in the VMN. Estradiol stimulates synthesis of progesterone, which in turn facilitates PACAP synthesis and activation of PAC1-short and -hop2 signaling critical for the induction of animal sexual receptivity.
PAC1 signaling is known to be required for physiological stress response as activation of PAC1 by PACAP is required for stress-induced CRH transcription in vivo and in vitro (Agarwal et al., 2005;Kageyama et al., 2007;Hammack et al., 2010b;Stroth and Eiden, 2010;Tsukiyama et al., 2011). The above studies suggest that PAC1 splice variants and their relative expression ratio might be involved in the regulation of body homeostasis including physiological responses to stressor challenges. A recent study performed in our lab, employed various stress paradigms in mouse and zebrafish to demonstrate that activation and termination of CRH transcription caused by stressful stimuli is regulated by an interrelationship of PAC1-null and hop1 isoforms (Amir-Zilberstein et al., 2012). Thus, the generation of PAC1-hop1 by alternative splicing leads to the termination of CRH transcription, normal activation of the hypothalamicpituitary-adrenal axis, and adaptive anxiety-like behavior (Amir-Zilberstein et al., 2012). We therefore suggested that alternative splicing of the hop1-cassette serves as an ON/OFF stress switch (Figure 6). PAC1 rs2267735 gene polymorphism is also associated with increased dark-enhanced startle (DES) in adult females but not males with PTSD (Ressler et al., 2011). Moreover, children of abused mothers show elevated DES and the same PAC1 gene polymorphism associated with PTSD risk in adult females is also associated with increased DES in these children (Jovanovic et al., , 2012. Notably, zebrafish larvae show a strong aversion to the dark side of a two-compartment light/dark arena and this place preference can be mitigated by anxiolytic drugs, such as Diazepam, indicating that this assay measures an anxiety-like behavior (Steenbergen et al., 2011;Schnorr et al., 2012). We found that during the recovery phase that follows a stressful challenge (osmotic shock), wild type larvae decrease their dark-avoidance time, a phenotype indicative of decreased stress-related anxiety, while larvae with depleted PAC1a-hop1 display delayed dark-avoidance recovery (Amir-Zilberstein et al., 2012). Thus, the delayed behavioral response of PAC1a-hop1-depleted embryos correlates with their respective failure to terminate crh and cortisol levels following stressors.

CONCLUSION
Alternative splicing is a major gene regulatory process involving cis-and trans-acting factors. PAC1 signaling controls a variety of cellular and physiological responses, such as differentiation, proliferation, cell cycle regulation, neurotransmitter, and hormone release and adaptation to stressful challenges. The PAC1 gene encompasses a relatively long genomic region, which consists of up to 18 exons and contains many putative splicing FIGURE 6 | A model illustrating the role of alternative splicing of PAC1 hop cassette that serves as an ON/OFF stress switch. In response to various stressors, the so called PAC1-null splice variant (i.e., no deletion in the N-terminal domain and no addition to the intracellular loops) modulate transcriptional activation of CRH and stress behaviors to adapt to the changes in homeostasis. Termination of CRH-mediated stress response is mediated by means of regulation of PAC1 gene splicing and inclusion of an altered exon (hop1) encoding to 28 amino acids of the third intracellular loop leading to the formation of the PAC1-hop1 splice variant. Generation of the PAC1-hop isoform terminates stress response by means yet to be uncovered (see text and Amir-Zilberstein et al., 2012). factors recognition sites that might be activated during different phases of neuronal activation. PAC1 receptor signaling can be fine-tuned by the generation of a set of alternatively spliced variants produced in a spatio-temporal manner. Splicingdependent alterations in PAC1 protein domains modify its ligand binding and signaling properties leading to a range of cellular activities. Although the physiological function(s) of the vast majority of the alternatively spliced PAC1 gene products is still unknown, recent studies have implicated certain PAC1 splice variants in the regulation of homeostatic processes such as adaptation to stressful challenges. In view of the recent association of PAC1 with PTSD risk, the regulation of PAC1 splicing and its underlying physiological outcomes might prove to be relevant to the etiology of some neurological and psychiatric disorders.