PAC1hop, null and hip receptors mediate differential signaling through cyclic AMP and calcium leading to splice variant-specific gene induction in neural cells

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP)-mediated activation of its G protein-coupled receptor PAC1 results in activation of the two G proteins Gs and Gq to alter second messenger generation and gene transcription in the nervous system, important for homeostatic responses to stress and injury. Heterologous expression of the three major splice variants of the rat PAC1 receptor, PAC1hop, null and hip, in neural NG108-15 cells conferred PACAP-mediated intracellular cAMP generation, while elevation of [Ca²⁺]_i occurred only in PAC1hop-, and to a lesser extent in PAC1null-expressing cells. Induction of vasoactive intestinal polypeptide (VIP) and stanniocalcin 1 (STC1), two genes potentially involved in PACAP's homeostatic responses, was examined as a function of the expressed PAC1 variant. VIP induction was greatest in PAC1hop-expressing cells, suggesting that a maximal transcriptional response requires combinatorial signaling through both cAMP and Ca²⁺. STC1 induction was similar for all three receptor splice variants and was mimicked by the adenylate cyclase activator forskolin, indicating that cAMP elevation is sufficient to induce STC1. The degree of activation of two different second messenger pathways appears to determine the transcriptional response, suggesting that cellular responses to stressors are fine-tuned through differential receptor isoform expression. Signaling to the VIP gene proceeded through cAMP and protein kinase A (PKA) in these cells, independently of the MAP kinase ERK1/2. STC1 gene induction by PACAP was dependent on cAMP and ERK1/2, independently of PKA. Differential gene induction via different cAMP dependent signaling pathways potentially provides further targets for the design of treatments for stress-associated disorders.

Introduction

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a peptide that was isolated from ovine hypothalamus based on its ability to stimulate cyclic adenosine 3′5′-monophosphate (cAMP) in rat anterior pituitary cells [33]. The mature peptide PACAP occurs in two C-terminally α-amidated forms, PACAP-27 and PACAP-38, with PACAP-27 being identical to the first 27 amino acids of PACAP-38 [34]. PACAP-27 has 68% sequence homology with vasoactive intestinal polypeptide (VIP), identifying PACAP as a member of the VIP-secretin-growth hormone releasing hormone-glucagon superfamily. The first 27 amino acids of PACAP have almost been completely preserved through vertebrate evolution, from fish to mammals, and are responsible for its biological activity [37], [50]. PACAP is widely expressed in the nervous system, e.g., the hypothalamus, cerebral cortex, amygdala, nucleus accumbens, hippocampus and cerebellum of the central nervous system, and in sensory neurons, sympathetic preganglionic neurons and parasympathetic pre- and postganglionic neurons of the peripheral nervous system. PACAP-38 is the predominant form expressed [4], [18], [20], [56].

PACAP binds to three G protein-coupled receptors (GPCRs), named VPAC1, VPAC2 and PAC1, which are members of the class B family of GPCRs. VPAC1 and VPAC2 bind VIP and PACAP with similar affinity, whereas PAC1 binds PACAP with high affinity and VIP with much lower affinity. PACAP receptors are abundantly expressed in the central nervous system, the anterior pituitary and adrenal gland [2], [23], [24], [28], [31], [45], [52], [53]. Several isoforms of the PACAP-preferring PAC1 receptor have been identified in vivo. These are generated through alternative splicing within two regions of the PAC1 gene: the N-terminus and the third intracellular loop (ic3). N-terminal variants result from deletions (21 or 57 amino acids) at the N-terminal extracellular domain affecting ligand binding and the relative potencies of the ligands in second messenger stimulation [9], [43]. Ic3 variants result from the presence or absence of different insertions at the C-terminal end of the loop, a domain thought to be crucial for interaction with G proteins. Each insertion, designated hip and hop, consists of an 84-bp cassette. The alternative use of two contiguous consensus splice acceptor sites at the 5′-end of the hop cassette generates hop1 and hop2. The hip cassette can be included together with the hop cassette to give rise to hiphop. The null form does not contain any insert. The hop cassette encodes a consensus motif for phosphorylation by protein kinase C (PKC) [54]. In the adrenal medulla the predominant PAC1 variant is PAC1hop, in the brain PAC1hop and null are abundantly expressed. PAC1 with a full-length N-terminus is the predominant form in the adult brain, whereas the embryonic brain expresses high levels of receptors containing a short N-terminus lacking 21 amino acids [15], [27], [38], [40], [46], [68].

All PACAP receptors regulate cAMP generation by coupling to adenylate cyclases (ACs) through Gs. Coupling to phospholipase Cβ (PLCβ), in contrast, varies among the different receptor sub-types. Regulation of inositol phosphate (IP) production by coupling to PLCβ through Gq is more efficacious in PAC1hop and null compared to hip receptors as assessed in non-neural heterologous cells [27], [46], [54]. The functional importance of Gs/AC- and Gq/PLCβ-mediated combinatorial signaling has been shown for sustained release of catecholamines (CAs) and neuropeptides from adrenomedullary chromaffin cells (CCs) [5], [16], [29], [49], [63], which predominantly express the PAC1hop receptor variant [38], [40]. In the adrenomedullary pheochromocytoma PC12 cell line [14] it has been shown that PAC1hop-activated sustained CA release proceeds through inositol-1,4,5-trisphosphate (IP₃)-mediated Ca²⁺ release from intracellular stores and store-operated Ca²⁺ entry (SOCE) [39], [58]. Moreover, the activation of two second messenger pathways, cAMP and Ca²⁺, seems to be required for maximal transcriptional stimulation of the neuropeptide VIP in CCs [17]. Stanniocalcin 1 (STC1) is another PACAP-regulated gene in CCs [1] with potentially neurotrophic functions [65], [66], [67]; signaling pathways regulating its neural expression, however, remain unidentified.

Although the different ic3 splice variants of PAC1 were first discovered almost 20 years ago, an understanding of second messenger production and gene induction mediated by these different variants in neural cells is still lacking. Therefore, we investigated the induction of the second messengers cAMP and Ca²⁺ as well as the PACAP target genes VIP and STC1 by the rat PAC1hop1, null and hip receptor variants with a full-length N-terminus in neural NG108-15 cells. The NG108-15 cell line is a neuroblastoma × glioma hybrid, not responding to PACAP endogenously, therefore providing an appropriate model system to study the different PAC1 splice variants separately introduced into a neural cell line. We demonstrate here that combinatorial signaling through cAMP and Ca²⁺, mediated uniquely by PAC1hop, is required for a full transcriptional response of the VIP gene. Cyclic AMP generation by either PAC1hop, null or hip is sufficient for induction of the gene encoding the neuroprotective protein STC1. Furthermore, two separate cAMP-dependent signaling pathways, activated by PACAP through PAC1hop, differentially regulate neural target genes. Our results provide evidence for the importance of differential expression of PAC1 splice variants and induction of second messenger pathways in shaping the PACAP-mediated transcriptional response in the nervous system.