Our research focuses on the biochemical signaling pathways regulated by muscarinic acetylcholine receptors (mAChR) and the Ras and Rho families of small GTPases. These proteins regulate important physiological processes in a variety of cell types, including contraction of smooth muscle and adhesion of epithelial cells. Abnormal signaling by these proteins may contribute to several diseases, including asthma, hypertension, cardiomyopathy, and cancer. The importance of these proteins in human health provides the driving force for our studies.
Nuclear localization signals in members of the Ras and Rho families of small GTPases
A major goal of our research is to understand how small GTPases in the Ras and Rho families regulate the cytoskeletal organization, adhesion, and proliferation of mammalian cells. It is generally accepted that members of the Ras and Rho families regulate critical cellular functions by interacting with proteins found in the cytoplasm of cells. However, we recently discovered that some members of the Rho family enter the cell nucleus, where these proteins may regulate nuclear functions. A specific amino acid sequence called the "nuclear localization signal" (or NLS) is required for many proteins to enter the nucleus. We observed that NLS sequences are present in the C-terminal regions of many Ras and Rho family members, and are evolutionarily conserved across several phyla. Our discovery of NLS sequences in small GTPases of the Ras and Rho families suggests that these proteins undergo nucleocytoplasmic shuttling, which is a previously unsuspected function of these small GTPases. The NLS may be required for the nuclear entry of small GTPases when they are associated with other proteins in complexes that are too large to passively diffuse through nuclear pores. The nuclear entry of these small GTPases may allow signals that are generated in the cytoplasm to be sensed in the nucleus. This ability to enter the nucleus expands the number of signaling pathways that are potentially regulated by these small GTPases, and may explain how these proteins can participate in a wide range of both normal and abnormal cellular responses.
Activation of small GTPases by Muscarinic Receptors
The M3 subtype of mAChR controls the activities of Rho family members in vascular smooth muscle cells. Thus, the M3 mAChR and these GTPases are intriguing therapeutic targets for diseases involving smooth muscle, including hypertension and asthma. A major goal of our research is to understand how the M3 mAChR regulates the activities of the small GTPases RhoA and Rac1. According to our model, the M3 mAChR-mediated activation of RhoA and Rac1 alters the organization of the actin/myosin cytoskeleton, resulting in increased cadherin- and integrin-mediated cell adhesion. To test this model, we transfect cDNAs encoding wildtype or mutant RhoA or Rac1 proteins into smooth muscle cells or other cell types expressing M3 mAChR. We use a variety of techniques, including fluorescence imaging and biochemical analysis of the expressed proteins, to characterize the effects of the transfected wildtype or mutant GTPases on the responses of the cells to mAChR activation. Using this approach, we demonstrated that RhoA participates in the M3 mAChR-mediated reorganization of the actin/myosin cytoskeleton, and Rac1 participates in the M3 mAChR-mediated increase in cadherin-mediated adhesion. Our finding that M3 mAChR activation regulates the activities of both phospholipase C-b1 and phospholipase C-b3 implicates these proteins in the M3 mAChR-mediated activation of RhoA and Rac1. We are currently identifying additional signaling molecules that contribute to the activation of RhoA and Rac1 by M3 mAChR.
Investigations of unique signaling pathways in small cell lung carcinoma
Although our research focuses on signaling by M3 mAChR and small GTPases in the cardiovascular system, we are also interested in the regulation and function of these signaling pathways in small cell lung carcinoma (SCLC) cells. We previously reported that activation of endogenous M3 mAChR diminishes the proliferation of SCLC cells, and induces both E-cadherin-mediated and integrin-mediated adhesion of SCLC cells. These mAChR-mediated events may decrease the metastatic potential of SCLC cells by decreasing their ability to break away from the primary tumor, migrate through connective tissues, and proliferate in secondary sites. Our studies implicate RhoA and Rac1 in these events, because RhoA and Rac1 regulate E-cadherin-mediated adhesion of SCLC cells. We recently found that RhoA expression is higher in SCLC cells than in other forms of lung cancer, suggesting that RhoA contributes to the high metastatic potential of SCLC cells. In other studies, we found that the expression of phospholipase C-b1 by SCLC cells may make these cells more resistant than other types of lung cancer to certain classes of chemotherapeutic agents. Our studies of SCLC signaling pathways will lead to a better understanding of how cell proliferation and adhesion are regulated in these cells, and may identify new approaches for the treatment of lung cancer.
Recent Publications
Lanning, C. C., Daddona, J. L., Ruiz-Velasco, R., Shafer, S. H., and C. L. Williams. 2004. The Rac1 C-terminal polybasic region regulates the nuclear localization and protein degradation of Rac1. J. Biol. Chem. (In Press).
Shafer, S. H. and C. L. Williams. 2004. Elevated Rac1 activity changes the M3 muscarinic acetylcholine receptor-mediated inhibition of proliferation to induction of cell death. Mol. Pharmacol. 65:1080-1091.
Williams, C. L. 2003. The polybasic region of Ras and Rho family members: A regulator of protein interactions and membrane association and a site of nuclear localization signal sequences. Cellular Signaling 15:1071-1080.
Lanning, C. C., Ruiz-Velasco, R., and C. L. Williams. 2003. Novel mechanism of the co-regulation of nuclear transport of SmgGDS and Rac1. Biol. Chem. 278:12495-12506.
Varker, K., S. Phelps, M. King, and C. L. Williams. 2003. The small GTPase RhoA has greater expression in small cell lung carcinoma than in non-small cell lung carcinoma and contributes to their unique morphologies. Int. J. Oncol. 22:671-681.
Shafer, S. H. and C. L. Williams. 2003. Non-small and small cell lung carcinoma cell lines exhibit cell type-specific sensitivity to edelfosine-induced cell death and different cell line-specific responses to edelfosine treatment. Int. J. Oncol. 23:389-400.
Williams, C. L. 2003. Muscarinic signaling in carcinoma cells. Life Sci. 72:2173-2182.
Ruiz-Velasco, R., C. C. Lanning, and C. L. Williams. 2002. The activation of Rac1 by M3 muscarinic acetylcholine receptors involves the translocation of Rac1 and IQGAP1 to cell junctions and changes in the composition of protein complexes containing Rac1, IQGAP1, and actin. J. Biol. Chem. 277:33081-33091.
Varker, K., and C. L. Williams. 2002. Involvement of the muscarinic acetylcholine receptor in inhibition of cell migration. Biochem. Pharm. 63:597-605.
Strassheim, D., S. Shafer, S. Phelps, and C. L. Williams. 2000. Small cell lung carcinoma exhibits greater phospholipase C-b1 expression and edelfosine resistance compared to non-small cell lung carcinoma. Cancer Res. 60:2730-2736.
Strassheim, D., R. Porter, S. Phelps, and C. L. Williams. 2000. Unique in vivo associations with SmgGDS and RhoGDI and different guanine nucleotide exchange activities exhibited by RhoA, dominant negative RhoAAsn19, and activated RhoAVal14. J. Biol. Chem. 275:6699-6702.
Strassheim, D., L. May, K. Varker, H. Puhl, S. Phelps, R. Porter, R. Aronstam, J. Noti, and C. L. Williams. 1999. M3 muscarinic acetylcholine receptors regulate cytoplasmic myosin by a process involving RhoA and requiring conventional protein kinase C isoforms. J. Biol.Chem. 274:18675-18685.