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Gonadotropin-releasing hormone receptor in the teleost Haplochromis burtoni: structure, location, and function - PubMed

Gonadotropin-releasing hormone receptor in the teleost Haplochromis burtoni: structure, location, and function

R R Robison et al. Endocrinology. 2001 May.

Abstract

GnRH acts via GnRH receptors (GnRH-R) in the pituitary to cause the release of gonadotropins that regulate vertebrate reproduction. In the teleost fish, Haplochromis burtoni, reproduction is socially regulated through the hypothalamus-pituitary-gonadal axis, making the pituitary GnRH-R a likely site of action for this control. As a first step toward understanding the role of GnRH-R in the social control of reproduction, we cloned and sequenced candidate GnRH-R complementary DNAs from H. burtoni tissue. We isolated a complementary DNA that predicts a peptide encoding a G protein-coupled receptor that shows highest overall identity to other fish type I GnRH-R (goldfish IA and IB and African catfish). Functional testing of the expressed protein in vitro confirmed high affinity binding of multiple forms of GNRH: Localization of GnRH-R messenger RNA using RT-PCR revealed that it is widely distributed in the brain and retina as well as elsewhere in the body. Taken together, these data suggest that this H. burtoni GnRH receptor probably interacts in vivo with all three forms of GNRH:

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Figures

Fig. 1
Fig. 1

A, GnRH-R nucleotide and predicted peptide sequences from H. burtoni. Transmembrane domains are shaded. Variant transcripts were found missing the bases between 1 and 2 and between 3 and 4 (positioned above the sequence here). Priming sites are indicated for the nested homology-based primers (underlined) and the H. burtoni-specific primers (italics). B, Comparison of predicted amino acid sequence homology among H. burtoni, several other teleost species, an amphibian (X. laevis), and a mammal, shown as percent identity. Sequence comparisons were made for the whole sequence, the amino extracellular tail alone, the whole sequence without the tails, and the carboxyl intracellular tail alone. C, Phylogenetic comparison of available cDNA sequences for GnRH-R. Note that nonmammalian sequences are separated into type I and type II (7). The H. burtoni GnRH-R reported here is type I, although an uncharacterized genomic DNA fragment (included here for comparison) aligns with type II sequences.

Fig. 2
Fig. 2

Distribution of GnRH-R in H. burtoni tissue assessed using RT-PCR. Primers based on H. burtoni GnRH-R were used on cDNA isolated from each tissue shown (see Materials and Methods). The first lane contains markers for 500 and 400 bp. Cb, Cerebellum; OT, optic tectum; Pit, pituitary; Hyp, hypothalamus; Tel, telencephalon; Control, RT without RNA, used as the PCR template.

Fig. 3
Fig. 3

Functional testing of the expressed cDNA for GnRH-R. Illustrated is the binding of GnRH and the production of IP in response to GnRH (see Materials and Methods). A, Whole cell competitive binding of 125I-[His5,D-Tyr6]GnRH after incubation with GnRH isoforms. B, Inositol phosphate in COS-1 cells after stimulation with GnRH.

Fig. 4
Fig. 4

A, Comparison of potencies among GnRH analogs that differ at a single residue, position 8. ED50 is plotted for four different GnRH agonists for IP production in COS-1 cells transiently transfected with the H. burtoni GnRH-R or the goldfish GnRH-R subtypes (6). B, Comparison of presumed binding regions of three GnRH-Rs by amino acids and by polarities of amino acids. The first extracellular loop (EC1) and the third extracellular loop (EC3) are compared for H. burtoni and the two goldfish GnRH-R forms.

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