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.