Genomic cloning and characterization of the human thrombin receptor gene. Structural similarity to the proteinase activated receptor-2 gene.
The seven-transmembrane segment thrombin receptor (TR) represents the prototype of a putative family of proteolytically cleaved receptors that may include the proteinase activated receptor-2. A panel of somatic cell hybrids retaining distinct portions of human chromosome 5 were used to establish that the human TR gene is present as a single-copy locus within the region 5q11.2 -->q13.3, confirming our previous localization using fluorescent in situ hybridization analysis. To further characterize the TR gene, overlapping clones from a human genomic library were isolated. Genomic analysis confirmed that the TR gene is of limited complexity, spanning approximately 27 kilobases and containing two exons separated by a large approximately 22-kilobase intron. The larger second exon contains the majority of the coding sequence and the thrombin cleavage site, remarkably similar to the organization of the proteinase activated receptor-2 gene in which the putative cleavage site is also contained within the large second exon. Primer extension analysis using two 30-mer oligonucleotide primers known to be contained within the first exon identified the predominant transcription initiation site 351 base pairs upstream from the initiator methionine in both human umbilical vein endothelial and human erythroleukemia cells. Sequence analysis of the 5'-flanking region revealed the TR promoter to be TATA-less, although nucleic acid motifspotentially involved in transcriptional gene regulation were evident and include a GATA motif, octamer enhancer sequences, AP-2-like sites, and Sp1 sites. These data provide evidence for remarkable similarity at the gene level between both proteolytically cleaved receptors described to date.
Schmidt
V A
VA
Department of Medicine, State University of New York at Stony Brook, New York 11794-8151, USA.
Vitale
E
E
Bahou
W F
WF
D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002872:Chromosome Deletion; D002874:Chromosome Mapping; D002895:Chromosomes, Human, Pair 5; D003001:Cloning, Molecular; D017931:DNA Primers; D005091:Exons; D015698:Genomic Library; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007438:Introns; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D044464:Receptor, PAR-2; D011956:Receptors, Cell Surface / Q000235:genetics*; D018179:Receptors, Thrombin / Q000096:biosynthesis* / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014158:Transcription, Genetic
D017931:DNA Primers; D044464:Receptor, PAR-2; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin