Amino acid, polyamine, and organocation (APC) transporters are secondary transporters that play essential roles in nutrient uptake, neurotransmitter recycling, ionic homeostasis, and regulation of cell volume. Here we present the crystal structure of apo-ApcT, a proton-coupled broad-specificity amino acid transporter, at 2.35 Å resolution. The structure contains 12 transmembrane helices, with the first 10 consisting of an inverted structural repeat of 5 transmembrane helices like LeuT. The ApcT structure reveals an inward-facing, apo state and an amine moiety of Lys158 located in a position equivalent to the Na2 ion of LeuT. We propose that Lys158 is central to proton coupled transport and that the amine group serves the same functional role as the Na2 ion in LeuT, thus demonstrating common principles amongst proton and sodium coupled transporters.Amino acid, polyamine and organocation (APC) transporters are members of a large family of secondary transport proteins that catalyze the uniport, symport and antiport of a broad range of substrates across the membrane bilayer (1). Present throughout the kingdom of life, APC transporters are integral to cellular physiology and in humans include multiple solute carrier (SLC) families ( Fig. 1A;table S1) (2). Canonical APC transporters include the SLC7 glutamate/ cystine antiporter (xCT), a sodium independent antiporter that exchanges extracellular cystine for intracellular glutamate(3,4). Cationic amino acid transporters (CATs) are also members of the SLC7 family and they supply arginine for nitric oxide synthesis, a major contributor to asthma pathogenesis (5). In cancer, the APC L-type amino acid transporter 1 provides amino acids required for tumor growth, is upregulated in tumor cells (6) and mediates the uptake of melphalin, a chemotherapy drug (7). Both cystinuria, the most common primary inherited aminoaciduria, and lysinuric protein intolerance are the consequence of mutations in SLC7 genes (8-10).The SLC12 family cation chloride cotransporters (CCCs) are subsumed under the APC mantle, are targets for several classes of therapeutic diuretics and are involved in active Cl -absorption in the kidney, blood pressure maintenance, and cell volume homeostasis (11); in the central nervous system, these transporters play an essential role in setting the resting chloride concentration and in γ-amino butyric acid (GABA)-and glycine-mediated neurotransmission (12). Other APC transporters participate in manifold biochemical processes in the nervous system, including the packaging of inhibitory neurotransmitters into synaptic vesicles (13) and the sodium or proton dependent symport of glutamine, a crucial step in the recycling of
G protein-coupled receptor kinase 2 (GRK2) plays a key role in the desensitization of G protein-coupled receptor signaling by phosphorylating activated heptahelical receptors and by sequestering heterotrimeric G proteins. We report the atomic structure of GRK2 in complex with Galphaq and Gbetagamma, in which the activated Galpha subunit of Gq is fully dissociated from Gbetagamma and dramatically reoriented from its position in the inactive Galphabetagamma heterotrimer. Galphaq forms an effector-like interaction with the GRK2 regulator of G protein signaling (RGS) homology domain that is distinct from and does not overlap with that used to bind RGS proteins such as RGS4.
The guanine nucleotide exchange factor p63RhoGEF is an effector of the heterotrimeric guanine nucleotide-binding protein (G protein) Galphaq and thereby links Galphaq-coupled receptors (GPCRs) to the activation of the small-molecular-weight G protein RhoA. We determined the crystal structure of the Galphaq-p63RhoGEF-RhoA complex, detailing the interactions of Galphaq with the Dbl and pleckstrin homology (DH and PH) domains of p63RhoGEF. These interactions involve the effector-binding site and the C-terminal region of Galphaq and appear to relieve autoinhibition of the catalytic DH domain by the PH domain. Trio, Duet, and p63RhoGEF are shown to constitute a family of Galphaq effectors that appear to activate RhoA both in vitro and in intact cells. We propose that this structure represents the crux of an ancient signal transduction pathway that is expected to be important in an array of physiological processes.
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