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Molecular cloning of the rat proteinase-activated receptor 4 (PAR4).

Hoogerwerf WA, Hellmich HL, Micci MA, Winston JH, Zou L, Pasricha PJ - BMC Mol. Biol. (2002)

Bottom Line: We have identified and characterized cDNA encoding the rat PAR4 homologue.PAR4 is expressed predominantly in the upper gastrointestinal tract.It is activated by trypsin, thrombin and its newly identified rat PAR4 specific activating peptide.

View Article: PubMed Central - HTML - PubMed

Affiliation: Enteric Neuromuscular Disorders and Pain Laboratory, Division of Gastroenterology and Hepatology, University of Texas Medical Branch, Galveston, TX, USA. wahooger@utmb.edu

ABSTRACT

Background: The proteinase-activated receptor 4 (PAR4) is a G-protein-coupled receptor activated by proteases such as thrombin and trypsin. Although activation of PAR4 has been shown to modulate rat gastrointestinal motility, the rat PAR4 sequence was unknown until now. This study aimed to identify the rat PAR4 cDNA.

Results: The cDNA coding for the rat PAR4 homologue was cloned from the duodenum. Northern blots demonstrated a 3.0 kb transcript in the duodenum. Protein homology with mouse and human counterparts was 90% and 75% respectively. PAR4 is expressed predominantly in the esophagus, stomach, duodenum and the spleen. When expressed in COS cells, PAR4 is activated by trypsin (1 nM), thrombin (50 nM), mouse PAR4 specific peptide (500 microM) and a putative rat PAR4 specific activating peptide (100 microM), as measured by intracellular Ca2+-changes.

Conclusions: We have identified and characterized cDNA encoding the rat PAR4 homologue. PAR4 is expressed predominantly in the upper gastrointestinal tract. It is activated by trypsin, thrombin and its newly identified rat PAR4 specific activating peptide.

No MeSH data available.


Ammo-acid sequence of rat PAR4 and alignment with mouse and human PAR4 (Genbank accession number AF080215, mouse, and AF080214, human). The putative rat PAR4 tethered ligand (GFPGKP) sequence has 3 amino-acids (GPG) in common with the human (GYPGQV) and 4 amino-acids (GPGK) with the mouse ligand (GYPGKF). Activating peptide sequences are printed bold.
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Figure 2: Ammo-acid sequence of rat PAR4 and alignment with mouse and human PAR4 (Genbank accession number AF080215, mouse, and AF080214, human). The putative rat PAR4 tethered ligand (GFPGKP) sequence has 3 amino-acids (GPG) in common with the human (GYPGQV) and 4 amino-acids (GPGK) with the mouse ligand (GYPGKF). Activating peptide sequences are printed bold.

Mentions: Primers, based upon the mouse PAR4 sequence, were designed to amplify the rat PAR4 coding region. We amplified a 1.1-kb fragment from rat duodenal RNA of which the nucleotide sequence shared 91 % and 82 % homology with mouse PAR4 and human PAR4 respectively. To establish the expression and corroborate the sequence of the PCR clone a rat duodenal cDNA library was screened. A partial clone was identified (0.7 kb), which contained part of the open reading frame (ORF) of the RT-PCR clone (nucleotides 596–1188) and a short segment of 3' untranslated region (nucleotides 1189–1323). To fill in the missing 5' end, 5' RACE was performed. This yielded a 0.6 kb fragment that represented the 5' end of the ORF (nucleotides 2–636). The ORF of the partial duodenal library clone and the 5' RACE fragments both shared 100% homology with the rat PAR4 PCR clone. Figure 1 shows the nucleotide sequence; figure 2 shows the deduced amino-acid sequence and its alignment with the human and mouse rat PAR4 homologue. The rat PAR4 nucleotide sequence is available in the GenBank under accession number AF310216.


Molecular cloning of the rat proteinase-activated receptor 4 (PAR4).

Hoogerwerf WA, Hellmich HL, Micci MA, Winston JH, Zou L, Pasricha PJ - BMC Mol. Biol. (2002)

Ammo-acid sequence of rat PAR4 and alignment with mouse and human PAR4 (Genbank accession number AF080215, mouse, and AF080214, human). The putative rat PAR4 tethered ligand (GFPGKP) sequence has 3 amino-acids (GPG) in common with the human (GYPGQV) and 4 amino-acids (GPGK) with the mouse ligand (GYPGKF). Activating peptide sequences are printed bold.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC88883&req=5

Figure 2: Ammo-acid sequence of rat PAR4 and alignment with mouse and human PAR4 (Genbank accession number AF080215, mouse, and AF080214, human). The putative rat PAR4 tethered ligand (GFPGKP) sequence has 3 amino-acids (GPG) in common with the human (GYPGQV) and 4 amino-acids (GPGK) with the mouse ligand (GYPGKF). Activating peptide sequences are printed bold.
Mentions: Primers, based upon the mouse PAR4 sequence, were designed to amplify the rat PAR4 coding region. We amplified a 1.1-kb fragment from rat duodenal RNA of which the nucleotide sequence shared 91 % and 82 % homology with mouse PAR4 and human PAR4 respectively. To establish the expression and corroborate the sequence of the PCR clone a rat duodenal cDNA library was screened. A partial clone was identified (0.7 kb), which contained part of the open reading frame (ORF) of the RT-PCR clone (nucleotides 596–1188) and a short segment of 3' untranslated region (nucleotides 1189–1323). To fill in the missing 5' end, 5' RACE was performed. This yielded a 0.6 kb fragment that represented the 5' end of the ORF (nucleotides 2–636). The ORF of the partial duodenal library clone and the 5' RACE fragments both shared 100% homology with the rat PAR4 PCR clone. Figure 1 shows the nucleotide sequence; figure 2 shows the deduced amino-acid sequence and its alignment with the human and mouse rat PAR4 homologue. The rat PAR4 nucleotide sequence is available in the GenBank under accession number AF310216.

Bottom Line: We have identified and characterized cDNA encoding the rat PAR4 homologue.PAR4 is expressed predominantly in the upper gastrointestinal tract.It is activated by trypsin, thrombin and its newly identified rat PAR4 specific activating peptide.

View Article: PubMed Central - HTML - PubMed

Affiliation: Enteric Neuromuscular Disorders and Pain Laboratory, Division of Gastroenterology and Hepatology, University of Texas Medical Branch, Galveston, TX, USA. wahooger@utmb.edu

ABSTRACT

Background: The proteinase-activated receptor 4 (PAR4) is a G-protein-coupled receptor activated by proteases such as thrombin and trypsin. Although activation of PAR4 has been shown to modulate rat gastrointestinal motility, the rat PAR4 sequence was unknown until now. This study aimed to identify the rat PAR4 cDNA.

Results: The cDNA coding for the rat PAR4 homologue was cloned from the duodenum. Northern blots demonstrated a 3.0 kb transcript in the duodenum. Protein homology with mouse and human counterparts was 90% and 75% respectively. PAR4 is expressed predominantly in the esophagus, stomach, duodenum and the spleen. When expressed in COS cells, PAR4 is activated by trypsin (1 nM), thrombin (50 nM), mouse PAR4 specific peptide (500 microM) and a putative rat PAR4 specific activating peptide (100 microM), as measured by intracellular Ca2+-changes.

Conclusions: We have identified and characterized cDNA encoding the rat PAR4 homologue. PAR4 is expressed predominantly in the upper gastrointestinal tract. It is activated by trypsin, thrombin and its newly identified rat PAR4 specific activating peptide.

No MeSH data available.