Compartmentalized accumulation of cAMP near complexes of multidrug resistance protein 4 (MRP4) and cystic fibrosis transmembrane conductance regulator (CFTR) contributes to drug-induced diarrhea.

Diarrhea is one of the most common adverse side effects observed in ∼7% of individuals consuming Food and Drug Administration (FDA)-approved drugs. The mechanism of how these drugs alter fluid secretion in the gut and induce diarrhea is not clearly understood. Several drugs are either substrates or inhibitors of multidrug resistance protein 4 (MRP4), such as the anti-colon cancer drug irinotecan and an anti-retroviral used to treat HIV infection, 3'-azido-3'-deoxythymidine (AZT). These drugs activate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated fluid secretion by inhibiting MRP4-mediated cAMP efflux. Binding of drugs to MRP4 augments the formation of MRP4-CFTR-containing macromolecular complexes that is mediated via scaffolding protein PDZK1. Importantly, HIV patients on AZT treatment demonstrate augmented MRP4-CFTR complex formation in the colon, which defines a novel paradigm of drug-induced diarrhea.

Förster resonance energy transfer - an approach to visualize the spatiotemporal regulation of macromolecular complex formation and compartmentalized cell signaling.

BACKGROUND: Signaling messengers and effector proteins provide an orchestrated molecular machinery to relay extracellular signals to the inside of cells and thereby facilitate distinct cellular behaviors. Formations of intracellular macromolecular complexes and segregation of signaling cascades dynamically regulate the flow of a biological process. SCOPE OF REVIEW: In this review, we provide an overview of the development and application of FRET technology in monitoring cyclic nucleotide-dependent signalings and protein complexes associated with these signalings in real time and space with brief mention of other important signaling messengers and effector proteins involved in compartmentalized signaling. MAJOR CONCLUSIONS: The preciseness, rapidity and specificity of cellular responses indicate restricted alterations of signaling messengers, particularly in subcellular compartments rather than globally. Not only the physical confinement and selective depletion, but also the intra- and inter-molecular interactions of signaling effectors modulate the direction of signal transduction in a compartmentalized fashion. To understand the finer details of various intracellular signaling cascades and crosstalk between proteins and other effectors, it is important to visualize these processes in live cells. Förster Resonance Energy Transfer (FRET) has been established as a useful tool to do this, even with its inherent limitations. GENERAL SIGNIFICANCE: FRET technology remains as an effective tool for unraveling the complex organization and distribution of various endogenous signaling proteins, as well as the spatiotemporal dynamics of second messengers inside a single cell to distinguish the heterogeneity of cell signaling under normal physiological conditions and during pathological events.

PKA and actin play critical roles as downstream effectors in MRP4-mediated regulation of fibroblast migration.

Multidrug resistance protein 4 (MRP4), a member of the ATP binding cassette transporter family, functions as a plasma membrane exporter of cyclic nucleotides. Recently, we demonstrated that fibroblasts lacking the Mrp4 gene migrate faster and contain higher cyclic-nucleotide levels. Here, we show that cAMP accumulation and protein kinase A (PKA) activity are higher and polarized in Mrp4(-/-) fibroblasts, versus Mrp4(+/+) cells. MRP4-containing macromolecular complexes isolated from these fibroblasts contained several proteins, including actin, which play important roles in cell migration. We found that actin interacts with MRP4, predominantly at the plasma membrane, and an intact actin cytoskeleton is required to restrict MRP4 to specific microdomains of the plasma membrane. Our data further indicated that the enhanced accumulation of cAMP in Mrp4(-/-) fibroblasts facilitates cortical actin polymerization in a PKA-dependent manner at the leading edge, which in turn increases the overall rate of cell migration to accelerate the process of wound healing. Disruption of actin polymerization or inhibition of PKA activity abolished the effect of MRP4 on cell migration. Together, our findings suggest a novel cAMP-dependent mechanism for MRP4-mediated regulation of fibroblast migration whereby PKA and actin play critical roles as downstream effectors.