Tunicate Gonadotropin-Releasing Hormone (GnRH) Peptides Selectively Activate Ciona intestinalis GnRH Receptors and the Green Monkey Type II GnRH Receptor

Abstract

In vertebrates, GnRH binds to its receptor and stimulates predominantly G(q/11)-mediated signal transduction in gonadotropes. However, little is known about the GnRH receptor and its signaling pathway in tunicates, a group that arose before the vertebrates. Although tunicates have had duplications of a few genes in the last 600 million years, the early vertebrates had duplications of the full genome. Also unknown is the nature of GnRH signaling in the tunicate, which lacks both a pituitary gland and sex steroids. However, we know that tunicates have GnRH peptides because we previously reported six GnRH peptides encoded within the tunicate genome of Ciona intestinalis. Here we clone and sequence cDNAs for four putative GnRH receptors from C. intestinalis. These are the only invertebrate GnRH receptors found to date. Each Ciona GnRH receptor was expressed in COS-7 cells, incubated with each of the six C. intestinalis GnRHs and assayed for a signaling response. GnRH receptors 1, 2, and 3 responded to Ciona GnRH peptides to stimulate intracellular cAMP accumulation. In contrast, only GnRH receptor 1 activated inositol phosphate turnover in response to one of the Ciona GnRHs. The green monkey type II GnRH receptor cDNA was tested as a comparison and a positive control. In conclusion, the four GnRH receptors encoded within the C. intestinalis genome were all transcribed into messenger RNA, but only three of the Ciona GnRH receptors were biologically active in our assays. The Ciona GnRH receptors almost exclusively activated the cAMP pathway.

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“…Instead, Ci-GnRHR-2 is responsive to t-GnRH-7, 8, 6 in this order of potency, while t-GnRH-3 and -5 specifically activate Ci-GnRHR-3 to a similar extent. Ci-GnRHR-4 exhibited neither elevation of intracellular calcium nor cAMP production (Tello et al, 2005). Instead, Ci-GnRHR-4 was shown to heterodimerize with Ci-GnRHR-1 and then potentiate the elevation of intracellular calcium, both calcium-dependent and -independent protein kinase C subtypes, and ERK phosphorylation in a ligand-selective fashion, verifying that CiGnRHR-4 serves as a protomer of GPCR heterodimers rather than a ligand-binding GPCR .…”

“…Ci-GnRHR-1 share 70%, 38%, and 36% sequence homology to Ci-GnRHR-2, -3, and -4, respectively, and all Ci-GnRHRs display approx. 30% sequence identity to human type-I GnRHR (Kusakabe et al, 2003;Tello et al, 2005). The phylogenetic analyses demonstrated that Ci-GnRHR-2, -3, and -4 are not homologs of vertebrate GnRHR subgroups but Ciona-specific paralogs of Ci-GnRHR-1 (Kusakabe et al, 2003;Tello et al, 2005).…”

“…Of particular significance is the distinct ligandselective production of second messengers mediated by Ci-GnRHRs. The elevation of intracellular calcium, which is typical of vertebrate and octopus GnRH and receptors (Millar 2005;Kanda et al, 2006), was only observed in administration of t-GnRH-6 to Ci-GnRHR-1, while other Ci-GnRHRs failed to stimulate the calcium elevation with any t-GnRHs (Tello et al, 2005). Instead, Ci-GnRHR-2 is responsive to t-GnRH-7, 8, 6 in this order of potency, while t-GnRH-3 and -5 specifically activate Ci-GnRHR-3 to a similar extent.…”

“…30% sequence identity to human type-I GnRHR (Kusakabe et al, 2003;Tello et al, 2005). The phylogenetic analyses demonstrated that Ci-GnRHR-2, -3, and -4 are not homologs of vertebrate GnRHR subgroups but Ciona-specific paralogs of Ci-GnRHR-1 (Kusakabe et al, 2003;Tello et al, 2005). Of particular significance is the distinct ligandselective production of second messengers mediated by Ci-GnRHRs.…”

“…This organization is closer to those of vertebrate gnrh genes than ci-gnrh-1 and -2. To date, four GnRH receptors, namely, Ci-GnRHR-1, -2, -3, and -4, have been identified in C. inetestinalis (Kusakabe et al, 2003;Tello et al, 2005). Ci-GnRHR-1 share 70%, 38%, and 36% sequence homology to Ci-GnRHR-2, -3, and -4, respectively, and all Ci-GnRHRs display approx.…”