Work in our lab is based on the simple premise that the formation of multiprotein signaling complexes at specialized plasma membrane microdomains increases the specificity and efficiency of signaling in polarized cells. We are specifically interested in understanding the formation of signaling complexes in epithelial cells and neurons and study the expression, localization, targeting and function of scaffolding and adaptor proteins in these cells.

One interesting feature of epithelial cells is that signals that originate at either the apical or basolateral cells surface do not always lead to detectable changes in the concentration of specific second messengers, although the cellular response is significantly altered. This suggests that receptors, signaling intermediates, and effectors are compartmentalized into regulatory complexes that increase the efficiency of signaling. In the last several years we described distinct apical and basolateral scaffolding complexes, and are currently studying the function of these complexes in airway, intestinal and kidney epithelial cell model systems. A hallmark feature of these scaffolding complexes is the presence of one or more proteins that contain PDZ domains, a protein interaction module involved in the clustering of receptors, signaling proteins, ion channels, and transporters at membrane specializations. We found that the apical membrane EBP50 protein complex could specifically associate with the COOH-terminus of the cystic fibrosis chloride channel (CFTR) and are very interested in understanding the composition and function of CFTR-associated scaffolding proteins in airway and intestinal epithelial cells.

We also found that EBP50 associates with YAP65, an adaptor protein known to associate in vitro with nonreceptor tyrosine kinases of the Src family. Now we are identifying extracellular signals that regulate the activity of apical membrane c-Yes, and are using biochemical approaches to identify substrates for tyrosine kinases in EBP50 protein complexes. In addition, in collaboration with Jack Stutts at the UNC CF Center, we recently found that Src family kinases inhibit the activity of the amiloride-sensitive Epithelial Na+ Channel (ENaC), a critical regulator of salt balance in the kidney and of mucociliary clearance in the airway. We are using a combination of approaches to elucidate the mechanism involved in the Src-mediated inhibition of ENaC including the yeast two-hybrid system and proteomic approaches to identify ENaC-associated proteins.

Protein kinase A is a critical regulator of CFTR activity in epithelial cells. Therefore, we want to identify proteins responsible for the compartmentalization of protein kinase A (PKA) at discrete plasma membrane domains in epithelial cells. These experiments are based on functional data generated in collaboration with Drs. Pingbo Huang and Jack Stutts at the UNC Cystic Fibrosis Center indicating that PKA-mediated regulation of CFTR requires that PKA be compartmentalized at the apical cell surface by association with an apical epithelial A-kinase anchoring protein (AKAP). AKAPs are a family of functionally related proteins that bind the regulatory (R) subunit of PKA with high affinity and target the kinase to specific subcellular organelles. Based on our functional studies in airway epithelial cells we screened cDNA expression libraries to clone novel AKAPs expressed in epithelial cells. We cloned several novel AKAPs and are studying their functions in epithelial cells and neurons.

We recently expanded our focus on epithelial cell biology to include the intestinal epithelium, and are studying the trafficking, regulation, and function of guanylyl cyclase C (GCC; also called the heat-stable enterotoxin receptor). GCC is the target for heat-stable enterotoxins that cause secretory diarrhea, a leading cause of infant death. We used mutagenesis to identify cytosolic signals important for the function and trafficking of GCC and used this information to generate probes for protein-interaction screens to identify proteins that associate with GCC. These screens led to the cloning of a novel epithelial-specific scaffolding protein that contains four PDZ domains. Based on its limited expression pattern (in kidney and intestine) and it domain structure we have named this novel protein IKEPP (Intestinal and Kidney Epithelial-Enriched PDZ Protein). We are currently studying the function of IKEPP in kidney and intestinal model systems and are using proteomic approaches to identify additional IKEPP-associated proteins.

Neurons are also highly polarized cells with axonal and somatodendritic compartments and highly localized signal transduction occurs in these specialized compartments. We are interested in scaffolding proteins involved in the targeting and compartmentalization of proteins in dendrites. We are studying the function of spinophilin, an actin-binding protein that we cloned in a yeast two-hybrid screen using the D2 dopamine receptor third cytoplasmic loop as the 'bait'. Spinophilin has multiple protein interaction domains including a binding site for protein phosphatase 1 and a single PDZ domain and is known to directly bind actin via an amino-terminal actin-binding domain. We are very interested in identifying proteins that associate with spinophilin and are using biochemical approached and two-hybrid screening to identify spinophilin-associated proteins. Once we have a better idea of the proteins that associate with spinophilin, we will study the function of the spinophilin - D2 dopamine receptor complex in neurons.

Mohler PJ, Kultgen PL, Stutts MJ, Milgram SL. Biochemical assays for
studying indirect interactions between CFTR and the cytoskeleton. Methods Mol Med. 70: 383 - 394, 2002.

Lee SW, Bonnah RA, Higashi DL, Atkinson JP, Milgram SL, So M. CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoea. J. Cell Biol. 156: 951 - 957, 2002.

Scott RO, Thelin WR, Milgram SL. A novel PDZ protein regulates the activity of guanylyl cyclase C, the heat-stable enterotoxin receptor, J Biol Chem, 277: 22934-22941, 2002

Barnes AP, Milgram SL. Signals from the X: signal transduction and X-linked mental retardation. Intl J Dev Neurosci, In Press 2002

Short DB, Trotter KW, Reczek D, Kreda S, Bretscher A, Boucher RC, Stutts MJ, and Milgram SL. An apical PDZ domain protein anchors CFTR to the cytoskeleton, J Biol Chem, 273:19797 - 19801, 1998.

Kachinsky AM, Froehner SC, and Milgram SL. A PDZ containing scaffold related to the dystrophin complex at the basolateral membrane of epithelial cells. J Cell Biol. 145:391-402, 1999.

Smith FD, Oxford GE, Milgram SL. Interaction of the D2 dopamine receptor third cytoplasmic loop with spinophilin, a protein phosphatase-1 interacting protein. J Biol. Chem., 274: 19894 -19900, 1999.