Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin
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S C Tsai1, R Adamik, M Tsuchiya
1Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation.
Insights
ADP-ribosylation factors (ARFs) are key regulators of cholera toxin activity. This study reveals developmental changes in ARF protein and mRNA levels in rat brain, correlating with neuronal maturation and synapse formation.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Cholera toxin utilizes ADP-ribosylation of guanine nucleotide-binding proteins for cellular effects.
- ADP-ribosylation factors (ARFs) are approximately 20-kDa proteins that allosterically activate the cholera toxin catalytic unit.
- ARFs play a crucial role in modulating cholera toxin-induced cellular responses.
Purpose of the Study:
- To investigate the presence and developmental regulation of ARF proteins and their corresponding mRNAs in mammalian brain.
- To correlate ARF expression patterns with neuronal maturation and synapse formation during postnatal development.
- To identify specific ARF gene products present in the rat brain.
Main Methods:
- Immunoblotting using rabbit antiserum against bovine brain ARF (sARF II) to detect ARF proteins in tissue extracts.
- Functional assays measuring cholera toxin-catalyzed ADP-ribosylation to assess ARF activity.
- Northern blot analysis of rat brain poly(A)+ RNA using cDNA and oligonucleotide probes specific for ARF genes.
- Nucleotide and amino acid sequence analysis of human and bovine ARF cDNAs.
Main Results:
- Two ARF-like proteins (sARF I and II) were detected in various species, with higher levels in brain tissue.
- In rat brain, sARF II predominated by the 10th postnatal day, and its levels correlated with functional cholera toxin assays.
- ARF 3 mRNA levels increased, while ARFs 2 and 4 mRNA levels decreased during postnatal development (days 2-27).
- ARF 1, 5, and 6 mRNA levels remained relatively unchanged during this period.
Conclusions:
- ARF proteins and their mRNAs exhibit distinct developmental regulation in the rat brain.
- The observed changes in ARF expression parallel key events in neuronal maturation and synapse formation.
- These findings highlight the dynamic role of ARFs in brain development and function.