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Identification of a serine protease which activates the mouse heart adenosine 5',5"',P1,P4-tetraphosphate receptor
1Department of Biological Sciences, Clemson University, South Carolina 29634-1903.
Abstract:
We have previously demonstrated that a serine protease dependent processing step is required for activation of the 30-kDa adenosine 5',5"',P1,P4-tetraphosphate (Ap4A) receptor. However, monoclonal antibodies (Mabs) against a 212-kDa polypeptide inhibit Ap4A binding to its receptor [Walker et al. (1993) Biochemistry 32, 1264-1269]. SDS-PAGE followed by autoradiography of [3H]diisopropylfluorophosphate (DIPF) covalently attached to membrane fractions reveals that the serine protease is the 212-kDa polypeptide or a proenzyme. Mabs against the 30-kDa Ap4A receptor are identified that inhibit Ap4A binding to its membrane receptor. These Mabs do not recognize the 212-kDa membrane protein but recognize four membrane proteins with molecular masses of 67, 55, 42, and 30 kDa. These data suggest that the precursor for the Ap4A receptor is a 67-kDa polypeptide which undergoes multiple cleavage events, at least one by the 212-kDa protein.
Insights
A 212-kDa serine protease processes the adenosine tetraphosphate (Ap4A) receptor. Monoclonal antibodies identified multiple precursor proteins, suggesting a complex maturation pathway for the Ap4A receptor.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Adenosine 5",5"-P1,P4-tetraphosphate (Ap4A) receptor activation requires serine protease processing.
- Previous studies indicated monoclonal antibodies (Mabs) against a 212-kDa polypeptide inhibit Ap4A binding.
Purpose of the Study:
- To identify the serine protease involved in Ap4A receptor activation.
- To characterize the precursor forms of the Ap4A receptor.
Main Methods:
- SDS-PAGE and autoradiography using [3H]diisopropylfluorophosphate (DIPF) to label serine proteases.
- Monoclonal antibody characterization against the Ap4A receptor and related proteins.
Main Results:
- The 212-kDa polypeptide was identified as the serine protease or its proenzyme.
- Mabs against the 30-kDa Ap4A receptor recognized proteins of 67, 55, 42, and 30 kDa.
- These Mabs did not recognize the 212-kDa protein.
Conclusions:
- The 67-kDa polypeptide is likely the precursor for the Ap4A receptor.
- Ap4A receptor maturation involves multiple cleavage events, including one mediated by the 212-kDa serine protease.