Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes - PubMed (original) (raw)
Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes
Z Guo et al. Proc Natl Acad Sci U S A. 1996.
Abstract
Lysophosphatidic acid (1-acyl-2-lyso-snglycero-3-phosphate, LPA) is a multifunctional lipid mediator found in a variety of organisms that span the phylogenetic tree from humans to plants. Although its physiological function is not clearly understood, LPA is a potent regulator of mammalian cell proliferation; it is one of the major mitogens found in blood serum. In Xenopus laevis oocytes, LPA elicits oscillatory Cl- currents. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways. Herein we report the identification of a complementary DNA from Xenopus that encodes a functional high-affinity LPA receptor. The predicted structure of this protein of 372 amino acids contains features common to members of the seven transmembrane receptor superfamily with a predicted extracellular amino and intracellular carboxyl terminus. An antisense oligonucleotide derived from the first 5-11 predicted amino acids, selectively inhibited the expression of the endogenous high-affinity LPA receptors in Xenopus oocytes, whereas the same oligonucleotide did not affect the low-affinity LPA receptor. Expression of the full-length cRNA in oocytes led to an increase in maximal Cl- current due to increased expression of the high-affinity LPA receptor, but activation of the low-affinity receptor was, again, unaffected. Oocytes expressing cRNA prepared from this clone showed no response to other lipid mediators including prostaglandins, leukotrienes, sphingosine 1-phosphate, sphingosylphosphorylcholine, and platelet-activating factor, suggesting that the receptor is highly selective for LPA.
Figures
Figure 1
(a) Antisense PAFR cRNA inhibits the endogenous LPA response in Xenopus oocytes. Oocytes were injected with increasing dilutions of a stock solution containing full-length PAFR antisense cRNA at 2 ng/nl. Responses to LPA were recorded up to 6 days later using a standard two-electrode voltage-clamp configuration with the membrane potential held at −60 mV. LPA was applied via superfusion at a concentration of 10 nM. Similar conditions were used throughout the experiments presented. Injection of PAFR antisense cRNA inhibited the LPA response in a dose-dependent fashion. (b) Design of degenerate oligonucleotides for PCR. Nucleotide sequence alignment of receptors for PAF (PAFR1, human; PAFR2, rat), thrombin (THR) (THR1, human; THR2, mouse), and ATP (ATPR1-4, human, chicken, turkey, and mouse, respectively) in the second and seventh transmembrane regions. Subscript numbers next to nucleotides in primers A and B represent their relative proportions used during synthesis. (c) PCR products isolated from the oocyte. Lanes of 1% agarose gels show the major amplification products obtained from the first PCR with primer A and the universal primer of the 3′-RACE kit (lane 1). Products of the nested PCR with primers A and B using the >1.2-kb template from the first reaction were cloned into the T-tail vector and three clones, representative of the three major PCR products, are shown in lanes 2–4 with different insert sizes (600–800 bp). (d) An 18-mer oligonucleotide (≈0.3 fmol per oocyte) derived from clone 71 selectively inhibits the endogenous LPA response, whereas it does not effect the cLPA response. The bar graph shows normalized mean currents to the low-affinity receptor-selective agonist cLPA (open bars, 1 μM) and the nonselective agonist LPA (solid bars, 10 nM). Injection of sense and random-sequence oligonucleotides did not affect the responses significantly.
Figure 2
(a) Nucleotide sequence of clone PSP24. Nucleotides underlined with a solid line represent part of the sequence encoding a putative signal peptide. Amino acid residues underlined indicate the predicted positions of the seven transmembrane regions. The dashed underline, in the amino acid sequence, marks the possible polyadenylylation site. Amino acids in boldface type and marked with asterisks represent possible phosphorylation sites, whereas those in italic type are potential N-glycosylation sites. The three asterisks mark the stop codon at the end of the open reading frame. (b) The putative amino acid sequence of the LPA receptor (LPAR) encoded by clone PSP24 shows sequence homology with different PAFRs (PAFR1, rat; PAFR2, guinea pig; PAFR3, human leukocyte; PAFR4, human heart; PAFR5, human granulocyte). Blocks drawn around the sequence mark conserved motifs. Asterisks under the sequence mark identical residues, whereas dots indicate conserved residues yielding a 17% sequence identity and an overall homology of 41% between these receptors.
Figure 3
(a) Representative responses to LPA, cLPA, and the other mediators used for primer design in control (upper trace) and PSP24 cRNA-injected (0.5 ng, lower trace) oocytes from the same frog. Notice that the cLPA response is practically identical in both cells, whereas the response to LPA is 2.5- to 3-fold higher (10 nM and 1 nM, respectively). None of the other mediators triggered an oscillatory response in either cell. (b) LPA dose–response relationship in sham-injected control (open circles,n = 4) and PSP24 cRNA-injected (solid squares,n = 5) oocytes. The responses in PSP24 cRNA-expressing oocytes exceeded those of the controls and showed an even more pronounced enhancement between 0.1 and 100 nM, contributing to the high-affinity receptor for LPA. (c) An antisense oligonucleotide designed from the sequence of amino acids 5–11 of clone PSP24 inhibits both the endogenous and the induced LPA response (n = 6 for all conditions). Injection of PSP24 cRNA at 0.5 ng per oocyte (solid bar) approximately doubled the response to 10 nM LPA in this experiment. Injection of ≈0.3 fmol (2.5 pg) of the 18-mer antisense oligonucleotide (hatched bar) reduced the endogenous response by 80%. To avoid the injection of double-stranded RNA that triggers the activation of nucleases, oocytes were injected a second time 2 days after the first injection with either PSP24 cRNA or the antisense oligonucleotide. A second injection of PSP24 cRNA (0.5 ng) into the oocytes (shaded bar) that were first injected with antisense oligonucleotide caused an ≈1.5-fold increase in the mean response over that of the distilled water-injected (“sham-injected”) controls. A second injection of the antisense oligonucleotide into oocytes that were first injected with PSP24 cRNA inhibited the response 73% below the endogenous response. All bars represent the mean response (±SEM) for six oocytes. (d) The increase in the LPA response is selective and dependent on the amount of PSP24 cRNA present. In this experiment, oocytes were injected with a 1- to 200-fold dilution of a stock solution of PSP24 cRNA (1 μg/μl, 50 nl per oocyte). A constant amount (0.3 fmol) of antisense cRNA was mixed with and coinjected with increasing dilutions of sense cRNA. Responses to 10 nM LPA and 1 μM cLPA were measured 4–6 days after injection. Traces are representative of four independent determinations (n = 4). Note that the increase in the LPA response decreased with the decreasing amount of PSP24 cRNA injected, whereas the cLPA response was virtually unaffected in all groups. Injection of 0.3 fmol of antisense cRNA, alone, caused a 45% reduction in the endogenous LPA response, whereas the cLPA response was virtually unaltered.
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