The human thrombin receptor and proteinase activated receptor‐2 genes are tightly linked on chromosome 5q13

Abstract

Summary. The thrombin receptor (TR) and proteinase activated receptor-2 (PAR-2) may represent the prototypes of an emerging family of cell-surface receptors that effect cell activation events mediated by serine proteases generated during inflammatory, fibrinolytic or haemostatic-regulated pathways. To further characterize the molecular genetics of these receptors, we have refined the genetic and physical mapping of both PAR-2 and TR. Utilization of two distinct radiation hybrid mapping panels with different levels of resolution demonstrated that both genes are tightly linked to the microsatellite markers D5S424, D5S1977, D5S2529 and D5S2596 (in order of decreasing LOD scores, from 13 . 7 for D5S424 to 7·7 for D5S2596). Physical mapping using yeast artificial chromosomes (YACs) and inversion field gel electrophoresis demonstrated that they are maximally separate by 90 kb. If the association of TR and PAR-2 genes resulted from a relatively recent gene duplication event from a common ancestral gene, these observations provide a general framework for the identification of gene transcripts representing alternative proteolytically activated receptors which may be clustered within this region of the human genome. These observations are especially relevant given recent evidence that murine and human platelets express alternative signalling mechanisms or receptors for thrombin.

The Human Proteinase-activated Receptor-3 (PAR-3) Gene

“…The separation of large DNA fragments on agarose gels was completed using an inversion field gel electrophoresis apparatus (Switchback TM pulse controller, Hoefer Scientific Instruments, San Francisco). Yeast artificial chromosomal (YAC) DNA was isolated as described previously (11), and human genomic fragments were separated from yeast chromosomes using a 1% agarose gel in 0.5 ϫ TBE (Tris borate-EDTA), run at 6.9 V/cm in 0.5 ϫ TBE buffer at 10°C with a pulse timer of 1-50 s and a forward/reverse ratio of 3:1 for 24 h. Gels were blotted onto nylon membranes (Schleicher & Schuell) and hybridized to 32 P-radiolabeled cDNA probes. Blots were washed at high (0.1 ϫ SSC, 0.1% SDS, 1 mM EDTA, pH 8.0, and 10 mM sodium phosphate at 68°C for 1 h) or low (0.1 ϫ SSC, 0.5% SDS, 1 mM EDTA, pH 8.0, 10 mM sodium phosphate at 55°C for 30 min) stringency and analyzed by autoradiography with Kodak XAR-5 film with an intensifying screen at Ϫ80°C for 3-10 days.…”

“…Radiation Hybrid (RH) Mapping Analysis-Genetic mapping within the PAR gene cluster was completed using both the Stanford G3 (ϳ500-kb resolution) and TNG3 (ϳ100-kb resolution) RH mapping panels (11,14). PCR screening was completed using the following PAR-3-specific oligonucleotide primers: PAR2064 (5Ј-3Ј: TCCATCCTTTCAC-CTACCGGG, bp 731-751) and PAR2065 (5Ј-3Ј: TAGCAGTAGATGAT-AAGCACA, bp 989 -969) (10).…”

“…PCR screening was completed using the following PAR-3-specific oligonucleotide primers: PAR2064 (5Ј-3Ј: TCCATCCTTTCAC-CTACCGGG, bp 731-751) and PAR2065 (5Ј-3Ј: TAGCAGTAGATGAT-AAGCACA, bp 989 -969) (10). PAR-1, PAR-2, and microsatellite-specific primers encompassing this region of the genome were as described previously (11). PCR conditions were optimized for individual primer pairs, but the thermal profile generally included a 1-min 95°C denaturation step, a 2-min 55°C annealing step, a 3-min 72°C primer extension step, with a final extension for 10 min at 72°C; after 25 cycles in a thermocycler (Coy Laboratory Products, Ann Arbor, MI), products were analyzed by Southern blot analysis in a 3% ethidium-stained agarose gel and scored as present or absent.…”

“…PAR-1-, PAR-2-, and PAR-3-specific oligonucleotide primers were used for determination of amplification (and retention) patterns using both G3 and TNG3 RH panels, followed by two-point linkage analysis using RH2PT (16). The centromeric/telomeric representation is a consensus from previous mapping data utilizing SHGC G3 RH panel; the position of D5S1977 relative to D5S424 and D5S2529 has not been confirmed in this laboratory and thus is presented in italics (11). Arbitrary distance measurements are expressed as cR_8000 (8,000 centiRad, the radiation dosage used for the generation of the G3 panel) or cR_50,000 (TNG3 panel).…”

How epithelial cells detect danger: aiding the immune response

“…These G proteins in turn directly regulate the activity of different intracellular enzymes (guanylylcyclase, adenylylcyclase, phospholipase C) or indirectly affect the activity of calcium-dependent enzymes by increasing intracellular Ca + + levels via calcium channels in the plasmamembrane and/or in the endoplasmatic recticulum. Three of the receptors (PAR-1, -2 and -3) are located in a small region (5q13) on chromosome 5 and share a two exon genomic organization with PAR-4 located on chromosome 19(p12) (60)(61)(62). A unique mechanism that uses the intrinsic enzyme activity of proteases to detect their presence underlies the activation of the PARs [ Fig.…”