A DNA sequence encoding a G-proteincoupled receptor was isolated from a mouse genomic library. The predicted protein is similar in structure to the thrombin receptor and has a similar activation mechanism. When expressed in Xenopus laevis oocytes, the receptor was activated by low concentrations of trypsin (EC 3.4.21.4) and by a peptide (SLIGRL) derived from the receptor sequence, but was not activated by thrombin (EC 3.4.21.5 It has been proposed that not all the cellular effects of thrombin can be ascribed to the newly cloned G-proteincoupled receptor (4), raising the possibility that there are subtypes of thrombin receptors. The cloning of thrombin receptors from Chinese hamster (5), rat (6), and mouse (GenBank accession no. L03529) has also been reported, but these receptors are all very similar to the human receptor, indicating that they represent the same kind of receptor in different species.When the human thrombin-receptor sequence was published, it was also speculated that this would be the first representative of a class of proteolytically activated receptors. However, Southern blot experiments with genomic DNA to probe for similar genes have proved unsuccessful, and no other reports have yet come forth to validate this speculation. Still, given the profusion of serine proteinases in various physiological systems-e.g., the blood-clotting cascade and the complement system, paired with the documented cellular effects displayed by, among others, plasmin and factor XIIa (7,8), this failure seems rather to speak of the limitations of current methodologies.The results presented here will shed some light on these issues. We report the cloning of a mouse genomic DNA sequence encoding a proteolytically activated receptor. § When expressed in frog oocytes, the receptor, which we provisionally have named proteinase activated receptor 2 (PAR-2), is activated by trypsin and by a synthetic peptide made from the receptor sequence. MATERIALS AND METHODSA mouse genomic library, cloned in the cosmid vector pTCF (9), was obtained from Rick A. Wetsel
We previously reported the molecular cloning of a mouse guanosine-nucleotide-binding-protein-coupled receptor similar to the thrombin receptor. Since the physiological agonist was unknown, the receptor was named proteinase-activated receptor 2. We describe here the cloning and functional expression of the gene encoding the corresponding human receptor. The gene is divided into two exons separated by about 14 kb intronic DNA. The deduced protein sequence is 397 amino acids long and 83% identical to the mouse receptor sequence. Within the extracellular amino terminus, the residues predicted to form the tethered agonist ligand differ between the two receptors; of the first six residues only four are conserved. At positions five and six, a lysine residue and a valine residue, respectively, have replaced arginine and leucine residues found in the mouse sequence. When the human receptor is expressed in Chinese hamster ovary cells, it can be activated by low nanomolar concentrations of the serine proteinase trypsin and by peptides made from the receptor sequence. Northem-blot analysis of receptor expression showed that the receptor transcript is widely expressed in human tissues with especially high levels in pancreas, liver, kidney, small intestine and colon. Moderate expression was detected in many organs but none in brain or skeletal muscle. By fluorescence in situ hybridization, the human proteinase-activated receptor 2 gene was mapped to chromosomal region 5q13, where, previously, the related thrombin receptor gene has been located.Keywords: chromosomal mapping; guanosione-nucleotide-binding-protein-coupled receptor; molecular cloning ; proteinase.The proteinase-activated receptor 2 (PAR-2) belongs to the large family of seven-transmembrane-region receptors that couple to guanosine-nucleotide-binding proteins. The physiological activator at this receptor is not known; apparently it is not activated by ordinary ligand binding but by proteolytic cleavage of its extracellular amino terminus [l]. The cleavage leaves the new amino terminus, a tethered ligand, free to interact with some other region of the receptor, and so effect its activation. PAR-2 shares this special mode of activation with the thrombin receptor, for which this mechanism was first described and has subsequently been studied extensively [2 -91.We have reported the cloning from genomic DNA of the mouse PAR-2 sequence [l]. When expressed in frog oocytes, the receptor could be activated with nanomolar concentrations of the serine proteinase trypsin but not with thrombin in doses up to 100 nM. The receptor could also be activated with a peptide (SLIGRL) corresponding to the postulated tethered ligand. From Southern-blot and Northern-blot data, it was judged that PAR-2 was present in the genome as a single copy gene and that, at least in the tissues analyzed, it was uniformly processed. Abbreviations. PAR-2, proteinase-activated receptor 2; CHO, Chinese hamster ovary ; FURA-2AM, FURA-2 acetoxymethyl ester.Enzyme. Bovine pancreatic trypsin type 111 (EC 3.4...
The proteinase-activated receptor 2 (PAR-2) belongs to the family of seven transmembrane region receptors, and, like the related thrombin receptor, it is activated by specific proteolytic cleavage of its extracellular amino terminus. It is not known which proteinase is the physiological activator of the PAR-2, but candidates can be found among the enzymes involved in the inflammatory cascade systems. Here, we have studied the effects of various mediators on the expression of the PAR-2 and the thrombin receptor in cultured human umbilical vein endothelial cells. Stimulation with the cytokines tumor necrosis factor ␣ or interleukin-1 ␣ as well as bacterial lipopolysaccharide elevated the expression of PAR-2 in a dose-dependent manner. The time course of induction after cytokine stimulation was similar to those published for the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. After 20 h of stimulation, PAR-2 mRNA and protein levels were increased to 5-10-fold basal values, and, in the continued presence of tumor necrosis factor ␣, PAR-2 mRNA expression was found to remain elevated for up to 4 days. In contrast, the thrombin receptor gene was not induced by any of these inflammatory mediators. The responses to phorbol ester treatment also differed between the two genes. Thrombin receptor mRNA levels decreased steadily up to 20 h, whereas PAR-2 mRNA levels first rose to about 3-fold basal values at 4 h before decreasing again. Cell surface protein levels of both receptors were decreased after 20 h of phorbol ester stimulation. Elevating intracellular cAMP levels by treatment with forskolin resulted in decreased expression of both receptors, and inhibition of cAMP degradation appeared to blunt the cytokine-induced increase in PAR-2 expression. The induction of the PAR-2 by cytokine treatment supports the concept of PAR-2 involvement in the acute inflammatory response.
Recently, a second member of the protease-activated receptor (PAR) family, named PAR-2, has been identified. Similar to the thrombin receptor, PAR-2 appears to be activated by proteolytic-mediated exposure of a "tethered ligand" sequence and can also be activated by the corresponding synthetic peptides. Similarities in the amino acid sequence of the receptors' tethered ligand sequences suggest that their respective agonist peptides might not be absolutely specific for their particular receptors. To test this, the receptor specificity of each agonist has been determined by measuring the responses of Xenopus oocytes expressing the thrombin receptor or PAR-2 to agonist peptides or enzymes. Thrombin receptors responded to thrombin, the human thrombin receptor-activating peptide SFLLRNP-NH2 (TRAP) (EC50 = 0.1 microM), and Xenopus TRAP, TFRIFD-NH2 (EC50 = 1 microM), but did not show any increase in calcium efflux over control levels with trypsin (50 nM) or PAR-2 agonist peptides (100 microM). Human and murine PAR-2 receptors responded comparably to human and murine PAR-2 agonist peptides (SLIGKVD and SLIGRL, respectively) (EC50 = 0.5-2.0 microM) and trypsin, but not to thrombin. PAR-2 was also found to be responsive to TRAP (EC50 = 1 microM) but was unresponsive to Xenopus TRAP (50 microM). Responses to additional peptide agonist analogs suggest that an amino-terminal serine is critical for PAR-2 agonist activity.
The complex of retinol with its carrier protein, retinol‐binding protein (RBP) has been crystallized and its three‐dimensional structure determined using X‐ray crystallography. Its most striking feature is an eight‐stranded up‐and‐down beta barrel core that completely encapsulates the retinol molecule. The retinol molecule lies along the axis of the barrel with the beta‐ionone ring innermost and the tip of the isoprene tail close to the surface.
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