Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor

Abstract

Recently we discovered a previously uncharacterized gene with the characteristics of a membrane progestin receptor (mPR) in a fish model, spotted seatrout. Here, we report the identification, cloning, and characteristics of other members of this hitherto unknown family of putative mPRs from several vertebrate species, including human, mouse, pig, Xenopus, zebrafish, and Fugu, with highly conserved nucleotide and deduced amino acid sequences and similar structures to the spotted seatrout mPR. The 13 vertebrate genes identified seem to belong to an unknown gene family. Phylogenetic analysis indicates these cDNAs comprise three distinct groups (named alpha, beta, and gamma) within this gene family. Structural analyses of the translated cDNAs suggest they encode membrane proteins with seven transmembrane domains. The transcript sizes of the human alpha, beta, and gamma putative mPR mRNAs varied from 2.8 to 5.8 kb and showed distinct distributions in reproductive, neural, kidney and intestinal tissues, respectively. Recombinant human alpha, gamma, and mouse beta proteins produced in an Escherichia coli expression system demonstrated high affinity (K(d) = 20-30 nM) saturable binding for progesterone. Further analysis of binding to the gamma-subtype revealed binding was specific for progestins and was displaceable, with rapid rates of association and dissociation (t(1/2) = 2-8 min). These results suggest this is a new family of steroid receptors unrelated to nuclear steroid receptors, but instead having characteristics of G protein-coupled receptors.

Figure 1

Unrooted phylogenetic tree (single most parsimonious tree, 932 steps; consistency index = 0.87; retention index = 0.86) showing the three clades of the putative mPRs. Branch lengths are proportional to the number of amino acid substitutions and bootstrap values (percentages); decay indexes are indicated on each branch.

Figure 2

Proposed model for insertion of the seatrout putative mPR in the plasma membrane based on hydrophilicity and charges of the amino acid residues analyzed by sosui (8). Each circle represents one amino acid residue. Residues identical in seatrout mPRα and the five other vertebrate mPRαs are indicated by the filled colored circles. Amino acid residues diagnostic of this clade are at positions 68 (T), 133 (A), 216 (S), 279 (Q), 310 (Y), 311 (E), 313 (L), and 329 (T) (filled black circles). Y shows a potential site of N-linked glycosylation, a cysteine residue (C). The proposed models for the mPRβs and mPRγs are very similar, with large N-terminal and second extracellular loop domains, and a short C-terminal domain (not shown). Amino acid residues diagnostic of mPRβ are at positions 6 (L), 11 (T), 22 (L), 89 (V), 102 (L), 114 (L), 138 (S), 167 (S), 203 (R), 204 (P), 205 (Y), 206 (P), 213 (R), 266 (V), 326 (S), 328 (F) from the N-terminal end of human mPRβ.

Figure 3

Amino acid sequence comparison between seatrout mPRα and the three putative human mPRs. Numbers above the box indicate percent sequence identity of amino acids in each domain between the fish mPR and human putative mPRs. Extracellular, solid gray; transmembrane, solid black; cytoplasmic domains, white.

Figure 4

(a) Dot blot hybridization of human mPR α (testicular), β (brain), and γ (kidney) mRNA probes with human multiple tissue arrays (CLONTECH). Only lanes displaying strong hybridizations with each of the probes are shown. Lanes 1–3 (various regions of CNS): b1 and g1, cerebral cortex; a2 and b2, cerebellum; e2, caudate nucleus; c3, thalamus; d3, pituitary gland; e3, spinal cord. Lane 4: regions of heart. Lanes 5 and 6 (gastrointestinal tract): h5, a6, and b6, colon. Lane 7 (kidney, hemopoietic, and lymphatic tissues): a7, kidney. Lane 8 (reproductive tissues): b8, placenta; d8, uterus; f8, testis; g8, ovary. Lane 9: liver and endocrine glands. Lane 10: lymphomas and carcinomas. Lane 11 (fetal tissues): c11, fetal kidney. (b–e) Northern hybridization of human α, β, and γ mRNA probes with human multiple tissue Northern blots (CLONTECH).

Figure 5

Steroid binding characteristics of recombinant mammalian proteins produced in E. coli. Saturation (a–c Insets) and Scatchard analyses of [³H]progesterone binding to recombinant mouse β (a), human α (b), and human γ (c) proteins. (d) Competition curves of steroid binding to the recombinant human mPRγ protein. Binding is expressed as a percentage of maximum specific binding. P4, progesterone; 17-P, 17α-hydroxyprogesterone; 20b-P, 20β-hydroxyprogesterone; E₂, estradiol-17β; T, testosterone; F, cortisol; RU486, synthetic antiprogestin. (e) Competition curves of binding of synthetic antihormones to the recombinant human mPRγ protein. Vit D: 1α, 25(OH)-vitamin D. Antiprogestins: ZK9, ZK98299; ZK1, ZK112983; ORG, ORG31710. Antiandrogen: Cim, cimetidine. Antiestrogen: ICI, ICI182,780. (f) Time course of association and dissociation of specific progesterone binding to human mPRγ.