The broad goal of the proposed study is to improve understanding of signal transduction pathways involved in B cell activation. This knowledge will provide important understanding of both normal physiology and unregulated cell growth. Abnormalities of signal transduction proteins have been identified in both cancer and immunodeficiencies. Bacterial toxins have been useful tools to dissect the function of G proteins, a group of signal transduction proteins. Using NAD as a source, bacterial toxins add adenosine diphosphate ribose (ADP-ribose) to proteins, a process called ADP-ribosylation. This modification can alter the function of signal transduction proteins in a manner that mimics their physiologic function. Based on his previous work, the applicant hypothesizes that cholera toxin ADP-ribosylates a non-Gs protein that regulates B cell activation. The goals are to identify this protein and to determine its physiologic function.
In Specific Aim 1, the ability of cholera toxin to ADP-ribosylate proteins in intact B cells will be used to block subsequent ADP-ribosylation of these proteins in vitro in the presence of [32P]NAD. While this reverse labeling technique has identified a single, non-Gs protein that is ADP-ribosylated by cholera toxin, other candidate proteins will be sought.
Specific Aim 2 is designed to develop the necessary tools to study the physiologic function of the ADP-ribosylated proteins. Following microsequencing, the sequence information will be used to clone the corresponding cDNA. At each step, sequence information will be compared to known amino acid and nucleic acid data bases 1) to help identify the protein or any functional motifs and 2) to determine which proteins to pursue for further analysis. Finally, antibodies will be generated.
Specific Aim 3 will begin to address the physiologic function of these proteins. Using antibodies, the investigator will determine whether and how these proteins are physiologically modulated by B cell stimulants, such as anti-Ig. In particular, he will determine whether these proteins are phosphorylated or dephosphorylated. If more than one candidate protein remains, these studies will establish which is the target protein. Conversely, selective activation of this protein by cholera toxin will permit him to explore the downstream effects that are specifically mediated by this protein.
