Limits...
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.


PAR4 mediated Ca2+ mobilization in transfected COS cells. (A) Rat thrombin 50 nM (1st arrow) followed by rat PAR4 AP 100 μM (2nd arrow), (B) Trypsin 1 nM (1st arrow) followed by rat PAR4 AP 100 μM (2nd arrow), (C) Mouse PAR4 AP 500 μM followed by repeat mouse PAR4 AP 500 μM, (D) Rat PAR4 AP 100 μM (1st arrow) followed by trypsin 1 nM (2nd and 3rd arrow), (E) Trypsin 1 nM (1st arrow) followed by rat PAR4 AP 100 μm (2nd arrow). Thr, thrombin; trp, trypsin; ms PAR4 AP, mouse PAR4 activating peptide.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC88883&req=5

Figure 5: PAR4 mediated Ca2+ mobilization in transfected COS cells. (A) Rat thrombin 50 nM (1st arrow) followed by rat PAR4 AP 100 μM (2nd arrow), (B) Trypsin 1 nM (1st arrow) followed by rat PAR4 AP 100 μM (2nd arrow), (C) Mouse PAR4 AP 500 μM followed by repeat mouse PAR4 AP 500 μM, (D) Rat PAR4 AP 100 μM (1st arrow) followed by trypsin 1 nM (2nd and 3rd arrow), (E) Trypsin 1 nM (1st arrow) followed by rat PAR4 AP 100 μm (2nd arrow). Thr, thrombin; trp, trypsin; ms PAR4 AP, mouse PAR4 activating peptide.

Mentions: Transfected cells responded to mouse PAR4 AP 500 μM, trypsin 1 nM, thrombin 50 nM and the putative rat PAR4 AP 100 μM (Figure 5). Control COS cells showed no response to any agonists tested (data not shown) and transfected cells failed to respond to the mouse control peptide. After incubation with mouse PAR4 AP, the cells did respond to a second incubation with the mouse PAR4 AP 500 μM, although the magnitude of the response was less. Rat PAR4 AP caused an increase in intracellular Ca2+ at 100 μM. Subsequent stimulation of the receptor with trypsin did not demonstrate an increase in intracellular Ca2+. When these agonists were applied in reversed order, the same observations were made: the receptor was activated by trypsin but subsequently did not respond to stimulation with rat PAR4 AP.


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)

PAR4 mediated Ca2+ mobilization in transfected COS cells. (A) Rat thrombin 50 nM (1st arrow) followed by rat PAR4 AP 100 μM (2nd arrow), (B) Trypsin 1 nM (1st arrow) followed by rat PAR4 AP 100 μM (2nd arrow), (C) Mouse PAR4 AP 500 μM followed by repeat mouse PAR4 AP 500 μM, (D) Rat PAR4 AP 100 μM (1st arrow) followed by trypsin 1 nM (2nd and 3rd arrow), (E) Trypsin 1 nM (1st arrow) followed by rat PAR4 AP 100 μm (2nd arrow). Thr, thrombin; trp, trypsin; ms PAR4 AP, mouse PAR4 activating peptide.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC88883&req=5

Figure 5: PAR4 mediated Ca2+ mobilization in transfected COS cells. (A) Rat thrombin 50 nM (1st arrow) followed by rat PAR4 AP 100 μM (2nd arrow), (B) Trypsin 1 nM (1st arrow) followed by rat PAR4 AP 100 μM (2nd arrow), (C) Mouse PAR4 AP 500 μM followed by repeat mouse PAR4 AP 500 μM, (D) Rat PAR4 AP 100 μM (1st arrow) followed by trypsin 1 nM (2nd and 3rd arrow), (E) Trypsin 1 nM (1st arrow) followed by rat PAR4 AP 100 μm (2nd arrow). Thr, thrombin; trp, trypsin; ms PAR4 AP, mouse PAR4 activating peptide.
Mentions: Transfected cells responded to mouse PAR4 AP 500 μM, trypsin 1 nM, thrombin 50 nM and the putative rat PAR4 AP 100 μM (Figure 5). Control COS cells showed no response to any agonists tested (data not shown) and transfected cells failed to respond to the mouse control peptide. After incubation with mouse PAR4 AP, the cells did respond to a second incubation with the mouse PAR4 AP 500 μM, although the magnitude of the response was less. Rat PAR4 AP caused an increase in intracellular Ca2+ at 100 μM. Subsequent stimulation of the receptor with trypsin did not demonstrate an increase in intracellular Ca2+. When these agonists were applied in reversed order, the same observations were made: the receptor was activated by trypsin but subsequently did not respond to stimulation with rat PAR4 AP.

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.