Paradoxical antagonism of PACAP receptor signaling by VIP in Xenopus oocytes via the type-C natriuretic peptide receptor☆
Introduction
Natriuretic peptides regulate central and peripheral body fluid balance and blood pressure. Three different peptides called ANP, BNP and CNP, have been identified in mammals [for review 1]. In fish, natriuretic peptides also exist and have been named ANP, BNP and VNP [2]. In frogs, ANP and BNP and CNP homologs have been isolated in Rana catesbeiana and Rana ridibunda [3], [4], [5]. Later, molecular cloning revealed that two isoforms of frog CNP, called CNP-I and CNP-II, coexist in R. catesbeiana [6]. Each of these peptides is generated by proteolytic cleavage of its respective prohormone, and possesses a characteristic ring shape due to an intramolecular disulfide bridge [1]. These hormones are mainly synthesized in the heart and brain, but they are also produced in other tissues such as rectal gland and skin in fish and frogs, respectively.
These peptides interact with four different receptors that have been identified by molecular cloning [1]. Two of them, called type A and B (NPR-A and NPR-B), contain guanylyl cyclase (GC) domains in their cytoplasmic C-terminal regions [7]. Activation of these receptors usually leads to an accumulation of intracellular cGMP. They differ in their affinity for various natriuretic peptides [8], [9]. ANP and BNP are the preferred ligands for mammalian and frog NPR-A [10], whereas CNP is the preferred ligand for NPR-B [11]. NPR-C and NPR-D have short intracellular tails devoid of intrinsic cyclase activity [12], [13]. They are characterized by a relative high affinity for all natriuretic peptides and a specific interaction with desANP4–23 a ring-deleted analogue. Because of its lack of selectivity, absence of GC activity, and capacity for ligand-mediated internalization, NPR-C has long been considered to be a clearance receptor, but strong evidence suggests that NPR-C has some specific signaling activities [for review 13]. Numerous studies indicate that NPR-C might be negatively or positively coupled to adenylyl cyclase (AC) [14], [15], or also positively to phospholipase C via βγ subunits [16]. NPR-D has been isolated only in eel [13], and shown not to differ from the eel C-type in its pharmacological features. However, it possesses an interesting sensitivity for HS-142-1 [17], a putative non-peptide antagonist specific for GC-containing natriuretic peptides receptors.
Natriuretic peptides' role in maintaining body fluid homeostasis may be crucial for species facing large environmental variations in salt concentrations such as fish and amphibians. Moreover, ANP-immunoreactivity has been observed in brain at early stages in Rana esculenta tadpoles, suggesting that natriuretic peptides might also play a role during brain development [18]. In mammals, the natriuretic peptide ligand/receptor systems may also participate in brain development [for review 19]. A direct action of natriuretic peptides on astrocyte and neuroblastoma cell proliferation has been observed in rodents [20], [21], [22]. Cardiac and vascular cell growth also appears to be regulated by natriuretic peptides in rodents [23], [24], [25] and chicks [26]. In mice, CNP overexpression or NPR-C gene inactivation [27], [28] triggered a pronounced overgrowth of bone skeleton, confirming the previous reports of a CNP-induced stimulation of cell proliferation in rat osteoblasts and chondrocytes [29], [30]. Furthermore, recent reports from our group and others strongly suggest that during development, protein kinase activation by natriuretic peptides may participate in the regulation of morphogenic actions of hedgehog signaling pathways [31], [32].
Here, we first report the cloning of the Xenopus natriuretic peptide type-C receptor (XNPR-C). Expression of this receptor by mRNA injection into oocytes was performed to study its pharmacological characteristics and potential signaling capabilities. This study demonstrated that NPR-C is indeed a member a VIP/PACAP receptor family. Second, we demonstrated that its negative coupling to cAMP production potentially counteracts PAC1 receptor activity, when the two receptors are co-expressed.
Section snippets
Xenopus brain library screening
A 450 bp cDNA was PCR-amplified from total RNA from fifty Xenopus tadpole heads using 5′-TGAGGACAGCGAAACCTGATT-3′ and 5′-GCAGGATTCTTCTAGGC CAC-3′ as primers. These were designed as consensus primers to fish and mammalian NPR-C. The amplified fragment was sequenced to confirm that it represented a NPR-C cDNA, then radiolabelled with [alpha-32P] dCTP using a random primer labelling kit (Invitrogen/Life Technologies) and used to screen a Xenopus brain lambda Ziplox cDNA library that was kindly
Isolation and analysis of a full-length cDNA encoding the Xenopus NPR-C
Using RT-PCR and consensus primers, we first isolated a 450 cDNA fragment that displayed high homology to NPR-C sequences of several species. Using this cDNA, we screened a lambda phage cDNA library and obtained two positive plaques. The Bluescript plasmids were excised using Ziplox rapid excision method. The insert sizes were 3.0 and 2.6 kb, respectively. Sequencing was performed in both directions. Only the 3.0 kb insert corresponded to a full-length cDNA, whereas the other was identical but
Discussion
Although NPR-C is best known for its clearance function, previous studies have suggested that NPR-C might also activate intracellular signaling pathways. Studies by the group of Anand-Srivastava [7], [13] and others [16] indicate that NPR-C activation results in inhibition of AC activity through pertussis toxin-sensitive Gi proteins. The present study confirms that Xenopus NPR-C also presents such a signaling pathway that elicited a pertussis toxin-sensitive inhibition of cAMP production.
Acknowledgments
We would thank A. Chao, J. Tam and W. Rodriguez for their helpful technical assistance. We are particularly grateful to Dr. E. De Robertis for providing us the cDNA library used in the present study and to Dr. R. Olcese for assistance in setting up the Xenopus oocyte microinjection technique. We also thank Dr. D. Alexandre for his helpful comments.
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This research was supported in part by NIH grants HD-06576, HD-0461 and HD-34475.
- 1
VL and ZH made equal contributions to the present work.
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Present address: INSERM U676, Hopital Robert Debre, 48 bd Serurier, F-75019 Paris, France.