Catherine H. Berlot

cAMP Regulates Ca2+-dependent Exocytosis of Lysosomes and Lysosome-mediated Cell Invasion by Trypanosomes

Ca2+-regulated exocytosis, previously believed to be restricted to specialized cells, was recently recognized as a ubiquitous process. In mammalian fibroblasts and epithelial cells, exocytic vesicles mobilized by Ca2+ were identified as lysosomes. Here we show that elevation in intracellular cAMP potentiates Ca2+-dependent exocytosis of lysosomes in normal rat kidney fibroblasts. The process can be modulated by the heterotrimeric G proteins Gs and Gi, consistent with activation or inhibition of adenylyl cyclase. Normal rat kidney cell stimulation with isoproterenol, a β-adrenergic agonist that activates adenylyl cyclase, enhances Ca2+-dependent lysosome exocytosis and cell invasion by Trypanosoma cruzi, a process that involves parasite-induced [Ca2+] i transients and fusion of host cell lysosomes with the plasma membrane. Similarly to what is observed for T. cruzi invasion, the actin cytoskeleton acts as a barrier for Ca2+-induced lysosomal exocytosis. In addition, infective stages of T. cruzi trigger elevation in host cell cAMP levels, whereas no effect is observed with noninfective forms of the parasite. These findings demonstrate that cAMP regulates lysosomal exocytosis triggered by Ca2+ and a parasite/host cell interaction known to involve Ca2+-dependent lysosomal fusion.

GS Activation Is Time-limiting in Initiating Receptor-mediated Signaling

To analyze individual steps of GS-linked signaling in intact cells, we used fluorescence resonance energy transfer (FRET)-based assays for receptor-G protein interaction, G protein activation, and cAMP effector activation. To do so, we developed a FRET-based sensor to directly monitor GS activation in living cells. This was done by coexpressing a Gαs mutant, in which a yellow fluorescent protein was inserted, together with cyan fluorescent protein-tagged Gβγ subunits and appropriate receptors in HEK293 cells. Together with assays for receptor activation and receptor-G protein interaction, it is possible to characterize large parts of the GS signaling cascade. When A2A-adenosine or β1-adrenergic receptors are coexpressed with GS in HEK293T cells, the receptor-GS interaction was on the same time scale as A2A receptor activation with a time constant of <50 ms. GS activation was markedly slower and around 450 ms with similar kinetics following activation of A2A- or β1-receptors. Taken together, our kinetic measurements demonstrate that the rate of GS activation limits initiation of GS-coupled receptor signaling.

Live Cell Imaging of Gs and the β2-Adrenergic Receptor Demonstrates That Both αs and β1γ7 Internalize upon Stimulation and Exhibit Similar Trafficking Patterns That Differ from That of the β2-Adrenergic Receptor

To visualize and investigate the regulation of the localization patterns of Gs and an associated receptor during cell signaling, we produced functional fluorescent fusion proteins and imaged them in HEK-293 cells. αs-CFP, with cyan fluorescent protein (CFP) inserted into an internal loop of αs, localized to the plasma membrane and exhibited similar receptor-mediated activity to that of αs. Functional fluorescent β1γ7 dimers were produced by fusing an amino-terminal yellow fluorescent protein (YFP) fragment to β1 (YFP-N-β1) and a carboxyl-terminal YFP fragment to γ7 (YFP-C-γ7). When expressed together, YFP-N-β1 and YFP-C-γ7 produced fluorescent signals in the plasma membrane that were not seen when the subunits were expressed separately. Isoproterenol stimulation of cells co-expressing αs-CFP, YFP-N-β1/YFP-C-γ7, and the β2-adrenergic receptor (β2AR) resulted in internalization of both fluorescent signals from the plasma membrane. Initially, αs-CFP and YFP-N-β1/YFP-C-γ7 stained the cytoplasm diffusely, and subsequently they co-localized on vesicles that exhibited minimal overlap with β2AR-labeled vesicles. Moreover, internalization of β2AR-GFP, but not αs-CFP or YFP-N-β1/YFP-C-γ7, was inhibited by a fluorescent dominant negative dynamin 1 mutant, Dyn1(K44A)-mRFP, indicating that the Gs subunits and β2AR utilize different internalization mechanisms. Subsequent trafficking of the Gs subunits and β2AR also differed in that vesicles labeled with the Gs subunits exhibited less overlap with RhoB-labeled endosomes and greater overlap with Rab11-labeled endosomes. Because Rab11 regulates traffic through recycling endosomes, co-localization of αs and β1γ7 on these endosomes may indicate a means of recycling specific αsβγ combinations to the plasma membrane.

A Highly Effective Dominant Negative αs Construct Containing Mutations That Affect Distinct Functions Inhibits Multiple Gs-coupled Receptor Signaling Pathways

To investigate the subcellular organization of receptor-G protein signaling pathways, a robust dominant negative αs mutant containing substitutions that alter distinct functions was produced and tested for its effects on Gs-coupled receptor activity in HEK-293 cells. Mutations in the α3β5 loop region, which increase receptor affinity, decrease receptor-mediated activation, and impair activation of adenylyl cyclase, were combined with G226A, which increases affinity for βγ, and A366S, which decreases affinity for GDP. This triple αs mutant can inhibit signaling to Gs from the luteinizing hormone receptor by 97% and from the calcitonin receptor by 100%. In addition, this αs mutant blocks all signaling from the calcitonin receptor to Gq. These results lead to two conclusions about receptor-G protein signaling. First, individual receptors have access to multiple types of G proteins in HEK-293 cell membranes. Second, different G protein α subunits can compete with each other for binding to the same receptor. This dominant negative αs construct will be useful for determining interrelationships among distinct receptor-G protein interactions in a wide variety of cells and tissues.

A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction

BackgroundThe jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins.ResultsWe tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, αs. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works.ConclusionThis technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.

Mutations at the domain interface of GSalpha impair receptor-mediated activation by altering receptor and guanine nucleotide binding.

G protein α subunits consist of two domains, a GTPase domain and a helical domain. Receptors activate G proteins by catalyzing replacement of GDP, which is buried between these two domains, with GTP. Substitution of the homologous αi2 residues for four αs residues in switch III, a region that changes conformation upon GTP binding, or of one nearby helical domain residue decreases the ability of αs to be activated by the β-adrenergic receptor and by aluminum fluoride. Both sets of mutations increase the affinity of αs for the β-adrenergic receptor, based on an increased amount of high affinity binding of the β-adrenergic agonist, isoproterenol. The mutations also decrease the rate of receptor-mediated activation and disrupt the ability of the β-adrenergic receptor to increase the apparent affinity of αs for the GTP analog, guanosine 5′-O-(3-thiotriphosphate). Simultaneous replacement of the helical domain residue and one of the four switch III residues with the homologous αi2 residues restores normal receptor-mediated activation, suggesting that the defects caused by mutations at the domain interface are due to altered interdomain interactions. These results suggest that interactions between residues across the domain interface are involved in two key steps of receptor-mediated activation, promotion of GTP binding and subsequent receptor-G protein dissociation.

Analysis of G Protein βγ Dimer Formation in Live Cells Using Multicolor Bimolecular Fluorescence Complementation Demonstrates Preferences of β1 for Particular γ Subunits

The specificity of G protein βγ signaling demonstrated by in vivo knockouts is greater than expected based on in vitro assays of βγ function. In this study, we investigated the basis for this discrepancy by comparing the abilities of seven β1γ complexes containing γ1, γ2, γ5, γ7, γ10, γ11, or γ12 to interact with αs and of these γ subunits to compete for interaction with β1 in live human embryonic kidney (HEK) 293 cells. βγ complexes were imaged using bimolecular fluorescence complementation, in which fluorescence is produced by two nonfluorescent fragments (N and C) of cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP) when brought together by proteins fused to each fragment. Plasma membrane targeting of αs-CFP varied inversely with its expression level, and the abilities of YFP-N-β1YFP-C-γ complexes to increase this targeting varied by 2-fold or less. However, there were larger differences in the abilities of the CFP-N-γ subunits to compete for association with CFP-C-β1. When the intensities of coexpressed CFP-C-β1CFP-N-γ (cyan) and CFP-C-β1YFP-N-γ2 (yellow) complexes were compared under conditions in which CFP-C-β1 was limiting, the CFP-N-γ subunits exhibited a 4.5-fold range in their abilities to compete with YFP-N-γ2 for association with CFP-C-β1. CFP-N-γ12 and CFP-N-γ1 were the strongest and weakest competitors, respectively. Taken together with previous demonstrations of a role for βγ in the specificity of receptor signaling, these results suggest that differences in the association preferences of coexpressed β and γ subunits for each other can determine which complexes predominate and participate in signaling pathways in intact cells.

Localization of the effector-specifying regions of Gi2alpha and Gqalpha.

Heterotrimeric G proteins transmit hormonal and sensory signals received by cell surface receptors to effector proteins that regulate cellular processes. Members of the highly conserved family of α subunits specifically modulate the activities of a diverse array of effector proteins. To investigate the determinants of α subunit-effector specificity, we localized the effector-specifying regions of αi2, which inhibits adenylyl cyclase, and αq, which stimulates phosphoinositide phospholipase C using chimeric α subunits. The chimeras were generated using an in vivo recombination method in Escherichia coli. The effector-specifying regions of both αi2 and αq were localized within the GTPase domain. An αq/αi2/αq chimera containing only 78 αi2 residues within the GTPase domain robustly inhibited adenylyl cyclase. This αi2 segment includes regions corresponding to two of the three regions of αs that activate adenylyl cyclase, but does not include any of the α subunit regions that switch conformation upon binding GTP. Replacement of the αq residues that comprise the helical domain with the homologous αi2 residues resulted in a chimeric α subunit that activated phospholipase C. Combined with previous studies of the effector-specifying residues of αs and αt, our results suggest that the effector specificity of α subunits is generally determined by the GTPase and not the helical domain.

Live Cell Analysis of G Protein β5 Complex Formation, Function, and Targeting

The G protein β5 subunit differs from other β subunits in having divergent sequence and subcellular localization patterns. Although β5γ2 modulates effectors, β5 associates with R7 family regulators of G protein signaling (RGS) proteins when purified from tissues. To investigate β5 complex formation in vivo, we used multicolor bimolecular fluorescence complementation in human embryonic kidney 293 cells to compare the abilities of 7 γ subunits and RGS7 to compete for interaction with β5. Among the γ subunits, β5 interacted preferentially with γ2, followed by γ7, and efficacy of phospholipase C-β2 activation correlated with amount of β5γ complex formation. β5 also slightly preferred γ2 over RGS7. In the presence of coexpressed R7 family binding protein (R7BP), β5 interacted similarly with γ2 and RGS7. Moreover, γ2 interacted preferentially with β1 rather than β5. These results suggest that multiple coexpressed proteins influence β5 complex formation. Fluorescent β5γ2 labeled discrete intracellular structures including the endoplasmic reticulum and Golgi apparatus, whereas β5RGS7 stained the cytoplasm diffusely. Coexpression of αo targeted both β5 complexes to the plasma membrane, and αq also targeted β5γ2 to the plasma membrane. The constitutively activated αo mutant, αoR179C, produced greater targeting of β5RGS7 and less of β5γ2 than did αo. These results suggest that αo may cycle between interactions with β5γ2 or other βγ complexes when inactive, and β5RGS7 when active. Moreover, the ability of β5γ2 to be targeted to the plasma membrane by α subunits suggests that functional β5γ2 complexes can form in intact cells and mediate signaling by G protein-coupled receptors.

Regulation of expression and function of scavenger receptor class B, type I (SR-BI) by Na+/H+ exchanger regulatory factors (NHERFs).

Background: SR-BI mediates selective delivery of lipoprotein-CE to the liver, adrenals, and gonads for product formation. Results: NHERFs interact with and down-regulate SR-BI protein levels to inhibit selective CE uptake and steroidogenesis. Conclusion: Two protein domains (PDZ and MERM) are required for NHERF1/2-mediated inhibition of SR-BI expression and function. Significance: This work reveals a novel mechanism of translational/posttranslational regulation of SR-BI. Scavenger receptor class B, type I (SR-BI) binds HDL and mediates selective delivery of cholesteryl esters (CEs) to the liver, adrenals, and gonads for product formation (bile acids and steroids). Because relatively little is known about SR-BI posttranslational regulation in steroidogenic cells, we examined the roles of Na+/H+ exchanger regulatory factors (NHERFs) in regulating SR-BI expression, SR-BI-mediated selective CE uptake, and steroidogenesis. NHERF1 and NHERF2 mRNA and protein are expressed at varying levels in model steroidogenic cell lines and the adrenal, with only low expression of PDZK1 (NHERF3) and NHERF4. Dibutyryl cyclic AMP decreased NHERF1 and NHERF2 and increased SR-BI mRNA expression in primary rat granulosa cells and MLTC-1 cells, whereas ACTH had no effect on NHERF1 and NHERF2 mRNA levels but decreased their protein levels in rat adrenals. Co-immunoprecipitation, colocalization, bimolecular fluorescence complementation, and mutational analysis indicated that SR-BI associates with NHERF1 and NHERF2. NHERF1 and NHERF2 down-regulated SR-BI protein expression through inhibition of its de novo synthesis. NHERF1 and NHERF2 also inhibited SR-BI-mediated selective CE transport and steroidogenesis, which were markedly attenuated by partial deletions of the PDZ1 or PDZ2 domain of NHERF1, the PDZ2 domain of NHERF2, or the MERM domains of NHERF1/2 or by gene silencing of NHERF1/2. Moreover, an intact COOH-terminal PDZ recognition motif (EAKL) in SR-BI is needed. Transient transfection of hepatic cell lines with NHERF1 or NHERF2 caused a significant reduction in endogenous protein levels of SR-BI. Collectively, these data establish NHERF1 and NHERF2 as SR-BI protein binding partners that play a negative role in the regulation of SR-BI expression, selective CE transport, and steroidogenesis.