(A) Genomic organization of mouse EP1 and PKN genes. The EP1 gene consists of 3 exons (black boxes) spanning approximately 7.2 kb. Shown are the translational ATG start site (nucleotides 2,700–2,702) and the premature stop codon and EcoRI site introduced in the mutant. PKN is antiparallel with EP1, and 2 alternative 3′ splice variants (arrows) are characterized by differences in the 3′ UTR. (B) Southern blot of EcoRI digest demonstrated successful incorporation of the EcoRI site into the EP1^–/– mouse. The radiolabeled EP1 probe recognized a 10-kb EcoRI fragment derived from the wild-type allele and a 5.8-kb fragment from the mutant allele. (C) Design of EP1^+/+ and EP1^–/– cDNA. The C795T mutation in knockout cDNA caused an in-frame R242X stop codon (CGA to TGA). Additionally, mutations C798G and G799A created a new EcoRI restriction site. (D) Secondary structure of EP1 receptor showing the site of truncation in the mutant receptor following the putative fifth membrane–spanning α-helix. (E) [Ca²⁺]_i signaling in fura-2–loaded HEK293 cells transfected with EP1^+/+ and EP1^–/– cDNA was determined using the 340/380 nm emission intensity ratio. PGE₂ (1 μM) significantly increased the ratio above basal values in cells transfected with EP1^+/+ expression vector but not in HEK293 cells transfected with EP1^–/–. (F) Autoradiogram in situ hybridization showed distribution of ³⁵S-labled riboprobes for EP1 antisense and PKN (EP1 sense) probes demonstrating nonoverlapping and inverse distribution patterns in mouse kidney. (G) Immunoprecipitation of EP1^+/+ and EP^–/– mouse kidney and brain lysates showed PKN expression was not altered in EP1^–/– mice.