Molecular characterizations of chicken and zebrafish prostanoid receptors and their implications on evolution of vertebrate prostanoidreceptor family

Abstract

Prostanoid receptors (PG-Rs: prostaglandin D, E, F, prostacyclin and

thromboxane receptors (DP, EP1-4, FP, IP and TP)) are known to mediate a diverse

range of biological responses, such as cardiovascular homeostasis, nociception and

reproduction, via binding to their respective ligands belonging to the five classes of

prostanoids (PGs: class D, E, F, I and thromboxane). The majority of these findings

were reported in mammals, and despite suggestive evidence provided by previous

pharmacological and physiological studies in non-mammalian vertebrates,

investigations on the mechanisms behind actions of PGs were impeded by the limited

information on their receptors.

In the present study, the full-length cDNAs of chicken (c-) and zebrafish

(z-) prostanoid receptors – cEP3, cFPs, zEP1s and zFP – were identified from

respective adult ovaries and their tissue distribution examined by RT-PCR. A novel

middle-truncated splice variant, cFPb, which lacks 107 amino acids between

transmembrane domains 4 and 6 but otherwise identical to cFPa was first identified.

Three isoforms of zEP1 – zEP1a, zEP1b, zEP1c – were found, which might have subfunctionalized

in their ligand binding and G protein coupling specificity, in addition to

differential tissue distribution. Using various luciferase reporter systems (pGL3-CRE,

pGL-NFAT-RE, pGL4-SRE), all the cloned receptors, except cFPb, were shown to

potentially couple to intracellular cAMP, Ca2+, and/or MAPK signaling pathways.

Owing to the proposed roles of PGs and its potential regulation by and/or

on EGFR ligands and gonadotropins in mammals and chicken, genes involved in

regulation of PG functions at various levels, including biosynthesis (COX1, COX2,

mPGES1, mPGES2 and cPGES), availability (PGT) and signaling (cEPs and cFPs),

were also characterized in granulosa cells during hen follicular development.

Lastly, using our experimental data and systematic sequence retrieval

from available databases, the PG receptor cascades from representative vertebrate

species were pooled and analysed using phylogenetic analyses and synteny studies.

Three putative clusters (IP-like, EP4-like and EP1-like cluster) were found in lamprey

genome; meanwhile, only one PG-R-like cluster was identified from the

Cephalochordate lancelet (amphioxus) genome. This concurs with the 1-2-4 rule

proposed in first round/second round (1R/2R) whole genome duplication in which the

missing lamprey cluster was presumably lost secondarily. With support from

conserved orthologs-localization, the four PG-R paralogs (proto-EP4, proto-IP/EP2/DP,

proto-TP/FP/EP1 & putative proto-EP3 genes) in the ancestral vertebrates might have

further diversified via either localized- (e.g. EP2 and DP) or chromosomal segmental

duplication (e.g. EP1, FP and TP) which resulted in the present array of vertebrate PGRs.

Additional paralogs (e.g. EP1 and EP4) were identified from fishes, by which

molecular dating coincide with and hint of their origins whence the ancient fishspecific

whole genome duplication (3R) occurred ~350 million years ago.

The present study offers the first glimpse and a better understanding of the

roles of the PG-Rs and presents a higher resolution to the evolutionary history of each

PG-R family member, consolidating that particular care has to be taken when studying

non-mammalian PG-R functions in which some members are absent or present in

multiples and propel the investigation of adaptational changes in the coding sequence

during evolution of vertebrate PG-Rs.