Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis

Arrestin binding to activated, phosphorylated G protein-coupled receptors (GPCRs) represents a critical step in regulation of light- and hormone-dependent signaling. Nonvisual arrestins, such as arrestin-2, interact with multiple proteins for the purpose of propagating and terminating signaling events. Using a combination of X-ray crystallography, molecular modeling, mutagenesis, and binding analysis, we reveal structural features of arrestin-2 that may enable simultaneous binding to phosphorylated receptor, SH3 domains, phosphoinositides, and beta-adaptin. The structure of full-length arrestin-2 thus provides a uniquely oriented scaffold for assembly of multiple, diverse molecules involved in GPCR signal transduction.     

Cell-to-cell transfer of glial proteins to the squid giant axon: The glia- neuron protein transfer hypothesis

The hypothesis that glial cells synthesize proteins which are transferred to adjacent neurons was evaluated in the giant fiber of the squid (Loligo pealei). When giant fibers are separated from their neuron cell bodies and incubated in the presence of radioactive amino acids, labeled proteins appear in the glial cells and axoplasm. Labeled axonal proteins were detected by three methods: extrusion of the axoplasm from the giant fiber, autoradiography, and perfusion of the giant fiber. This protein synthesis is completely inhibited by puromycin but is not affected by chloramphenicol. The following evidence indicates that the labeled axonal proteins are not synthesized within the axon itself. (a) The axon does not contain a significant amount of ribosomes or ribosomal RNA. (b) Isolated axoplasm did not incorporate [(3)H]leucine into proteins. (c) Injection of Rnase into the giant axon did not reduce the appearance of newly synthesized proteins in the axoplasm of the giant fiber. These findings, coupled with other evidence, have led us to conclude that the adaxonal glial cells synthesize a class of proteins which are transferred to the giant axon. Analysis of the kinetics of this phenomenon indicates that some proteins are transferred to the axon within minutes of their synthesis in the glial cells. One or more of the steps in the transfer process appear to involve Ca++, since replacement of extracellular Ca++ by either Mg++ or Co++ significantly reduces the appearance of labeled proteins in the axon. A substantial fraction of newly synthesized glial proteins, possibly as much as 40 percent, are transferred to the giant axon. These proteins are heterogeneous and range in size from 12,000 to greater than 200,000 daltons. Comparisons of the amount of amino acid incorporation in glia cells and neuron cell bodies raise the possibility that the adaxonal glial cells may provide an important source of axonal proteins which is supplemental to that provided by axonal transport from the cell body. These findings are discussed with reference to a possible trophic effect of glia on neurons and metabolic cooperation between adaxonal glia and the axon.     

Magnetic resonance studies of the interaction of divalent metal cations with 2,3-bisphosphoglycerate

The binding of Mg²⁺ to intracellular 2,3-bisphosphoglycerate in the human red blood cell is significant to the function of the cell. We have studied interactions of Mg²⁺ and Mn²⁺ with 2,3-bisphosphoglycerate by magnetic resonance spectroscopy. The results of this study reveal the presence of two independent divalent metal cation binding sites of similar affinity (K_D = 3.0 ± 0.5 mM) in the 2,3-bisphosphoglycerate molecule, one on each phosphoryl group, contrary to the assumption of one metal ion binding site made in the previous literature. Over the range of their intracellular concentrations, ATP and ADP, however, possess only one metal ion site in spite of the presence of multiple phosphoryl groups. These results are consistent with the chemistry of metal-chelation which requires the formation of 5- or 6-membered rings for the stability of chelate structures.     

Five members of a novel Ca(2+)-binding protein (CABP) subfamily with similarity to calmodulin

Five members of a novel Ca(2+)-binding protein subfamily (CaBP), with 46-58% sequence similarity to calmodulin (CaM), were identified in the vertebrate retina. Important differences between these Ca(2+)-binding proteins and CaM include alterations within their second EF-hand loop that render these motifs inactive in Ca(2+) coordination and the fact that their central alpha-helixes are extended by one alpha-helical turn. CaBP1 and CaBP2 contain a consensus sequence for N-terminal myristoylation, similar to members of the recoverin subfamily and are fatty acid acylated in vitro. The patterns of expression differ for each of the various members. Expression of CaBP5, for example, is restricted to retinal rod and cone bipolar cells. In contrast, CaBP1 has a more widespread pattern of expression. In the brain, CaBP1 is found in the cerebral cortex and hippocampus, and in the retina this protein is found in cone bipolar and amacrine cells. CaBP1 and CaBP2 are expressed as multiple, alternatively spliced variants, and in heterologous expression systems these forms show different patterns of subcellular localization. In reconstitution assays, CaBPs are able to substitute functionally for CaM. These data suggest that these novel CaBPs are an important component of Ca(2+)-mediated cellular signal transduction in the central nervous system where they may augment or substitute for CaM.     

Mutational analysis of Gbetagamma and phospholipid interaction with G protein-coupled receptor kinase 2

Agonist-dependent regulation of G protein-coupled receptors is dependent on their phosphorylation by G protein-coupled receptor kinases (GRKs). GRK2 and GRK3 are selectively regulated in vitro by free Gbetagamma subunits and negatively charged membrane phospholipids through their pleckstrin homology (PH) domains. However, the molecular binding determinants and physiological role for these ligands remain unclear. To address these issues, we generated an array of site-directed mutants within the GRK2 PH domain and characterized their interaction with Gbetagamma and phospholipids in vitro. Mutation of several residues in the loop 1 region of the PH domain, including Lys-567, Trp-576, Arg-578, and Arg-579, resulted in a loss of receptor phosphorylation, likely via disruption of phospholipid binding, that was reversed by Gbetagamma. Alternatively, mutation of residues distal to the C-terminal amphipathic alpha-helix, including Lys-663, Lys-665, Lys-667, and Arg-669, resulted in decreased responsiveness to Gbetagamma. Interestingly, mutation of Arg-587 in beta-sheet 3, a region not previously thought to interact with Gbetagamma, resulted in a specific and profound loss of Gbetagamma responsiveness. To further characterize these effects, two mutants (GRK2(K567E/R578E) and GRK2(R587Q)) were expressed in Sf9 cells and purified. Analysis of these mutants revealed that GRK2(K567E/R578E) was refractory to stimulation by negatively charged phospholipids but bound Gbetagamma similar to wild-type GRK2. In contrast, GRK2(R587Q) was stimulated by acidic phospholipids but failed to bind Gbetagamma. In order to examine the role of phospholipid and Gbetagamma interaction in cells, wild-type and mutant GRK2s were expressed with a beta(2)-adrenergic receptor (beta(2)AR) mutant that is responsive to GRK2 phosphorylation (beta(2)AR(Y326A)). In these cells, GRK2(K567E/R578E) and GRK2(R587Q) were largely defective in promoting agonist-dependent phosphorylation and internalization of beta(2)AR(Y326A). Similarly, wild-type GRK2 but not GRK2(K567E/R578E) or GRK2(R587Q) promoted morphinedependent phosphorylation of the mu-opioid receptor in cells. Thus, we have (i) identified several specific GRK2 binding determinants for Gbetagamma and phospholipids, and (ii) demonstrated that Gbetagamma binding is the limiting step for GRK2-dependent receptor phosphorylation in cells.     

Regulation of cysteinyl leukotriene type 1 receptor internalization and signaling

Cysteinyl leukotrienes activate the cysteinyl leukotriene type 1 receptor (CysLT1R) to regulate numerous cell functions important in inflammatory processes and diseases such as asthma. Despite its physiologic importance, no studies to date have examined the regulation of CysLT1R signaling or trafficking. We have established model systems for analyzing recombinant human CysLT1R and found regulation of internalization and signaling of the CysLT1R to be unique among G protein-coupled receptors. Rapid and profound LTD4-stimulated internalization was observed for the wild type (WT) CysLT1R, whereas a C-terminal truncation mutant exhibited impaired internalization yet signaled robustly, suggesting a region within amino acids 310-321 as critical to internalization. Although overexpression of WT arrestins significantly increased WT CysLT1R internalization, expression of dominant-negative arrestins had minimal effects, and WT CysLT1R internalized in murine embryonic fibroblasts lacking both arrestin-2 and arrestin-3, suggesting that arrestins are not the primary physiologic regulators of CysLT1Rs. Instead, pharmacologic inhibition of protein kinase C (PKC) was shown to profoundly inhibit CysLT1R internalization while greatly increasing both phosphoinositide (PI) production and calcium mobilization stimulated by LTD4 yet had almost no effect on H1 histamine receptor internalization or signaling. Moreover, mutation of putative PKC phosphorylation sites within the CysLT1R C-tail (CysLT1RS(313-316)A) reduced receptor internalization, increased PI production and calcium mobilization by LTD4, and significantly attenuated the effects of PKC inhibition. These findings characterized the CysLT1R as the first G protein-coupled receptor identified to date in which PKC is the principal regulator of both rapid agonist-dependent internalization and rapid agonist-dependent desensitization.