Stochastic receptor expression determines cell fate upon interferon treatment

Type I interferons trigger diverse biological effects by binding a common receptor, composed of IFNAR1 and IFNAR2. Intriguingly, while the activation of an antiviral state is common to all cells, antiproliferative activity and apoptosis affect only part of the population, even when cells are stimulated with saturating interferon concentrations. Manipulating receptor expression by different small interfering RNA (siRNA) concentrations reduced the fraction of responsive cells independent of the interferon used, including a newly generated, extremely tight-binding variant. Reduced receptor numbers increased 50% effective concentrations (EC(50)s) for alpha interferon 2 (IFN-α2) but not for the tight-binding variant. A correlation between receptor numbers, STAT activation, and gene induction is observed. Our data suggest that for a given cell, the response is binary (+/-) and dependent on the stochastic expression levels of the receptors on an individual cell. A low number of receptors suffices for antiviral response and is thus a robust feature common to all cells. Conversely, a high number of receptors is required for antiproliferative activity, which allows for fine-tuning on a single-cell level.     

The phosphorylation and tyrosine kinase activity of an internalized complex of epidermal growth factor and its receptor

A study was made of the functional state of the epidermal growth factor (EGF)--receptor complexes in A-431 cells. Conditions of surface bound EGF extraction were selected which allow to consider the intracellular EGF--receptor complexes only. A procedure of high efficient and specific immunoprecipitation of tyrosyl-phosphorylated EGF receptors was developed. It is shown that the dissociation of EGF--receptor complexes leads to receptor dephosphorylation due to a rapid and reversible inactivation of EGF receptor tyrosine kinase. The internalized receptor is found to be tyrosyl-phosphorylated and to retain tyrosine kinase for at least an hour after the internalization. The dynamics of dissociation, degradation and dephosphorylation of EGF--receptor complexes has been estimated. The rates of these processes prove to be almost negligible for the first 2.5 hours after internalization.     

The third intracellular loop of alpha 2-adrenergic receptors determines subtype specificity of arrestin interaction

Nonvisual arrestins (arrestin-2 and -3) serve as adaptors to link agonist-activated G protein-coupled receptors to the endocytic machinery. Although many G protein-coupled receptors bind arrestins, the molecular determinants involved in binding remain largely unknown. Because arrestins selectively promote the internalization of the alpha(2b)- and alpha(2c)-adrenergic receptors (ARs) while having no effect on the alpha(2a)AR, here we used alpha(2)ARs to identify molecular determinants involved in arrestin binding. Initially, we assessed the ability of purified arrestins to bind glutathione S-transferase fusions containing the third intracellular loops of the alpha(2a)AR, alpha(2b)AR, or alpha(2c)AR. These studies revealed that arrestin-3 directly binds to the alpha(2b)AR and alpha(2c)AR but not the alpha(2a)AR, whereas arrestin-2 only binds to the alpha(2b)AR. Truncation mutagenesis of the alpha(2b)AR identified two arrestin-3 binding domains in the third intracellular loop, one at the N-terminal end (residues 194-214) and the other at the C-terminal end (residues 344-368). Site-directed mutagenesis further revealed a critical role for several basic residues in arrestin-3 binding to the alpha(2b)AR third intracellular loop. Mutation of these residues in the holo-alpha(2b)AR and subsequent expression in HEK 293 cells revealed that the mutations had no effect on the ability of the receptor to activate ERK1/2. However, agonist-promoted internalization of the mutant alpha(2b)AR was significantly attenuated as compared with wild type receptor. These results demonstrate that arrestin-3 binds to two discrete regions within the alpha(2b)AR third intracellular loop and that disruption of arrestin binding selectively abrogates agonist-promoted receptor internalization.     

Cdx2 determines the fate of postnatal intestinal endoderm

Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2-negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.     

Factors determining the ultimate fate of a plastic surgery applicant

Plastic surgery residency program directors are frequently interested in predictors of future career direction in their applicants. Many programs strive to train leaders in academic plastic surgery. To determine what factors may predict the ultimate fate of graduating plastic surgery residents, the authors reviewed the application files of 33 former residents from a single, major plastic surgery training program. The data from 29 residents were available for analysis. Nearly half of the residents graduating from the plastic surgery training program went into private practice. Two factors, the number of years taken off for research before entering the plastic surgery residency and the presence of children, were found to be indicative of a candidate's future career path. Of particular note, there was no difference between academic graduates and nonacademic graduates with regard to their intentions in their letters of recommendation and personal statements. This information is useful to both academic program directors and resident applicants.     

The dynamic nature of the Golgi complex

The intracellular transport of newly synthesized G protein of vesicular stomatitis virus is blocked at 20 degrees C and this spanning membrane glycoprotein accumulates in the last Golgi compartment, the trans Golgi-network (TGN). Previous morphological evidence suggested that the TGN enlarged significantly under this condition. In the present study we have used stereological procedures to estimate the volume and surface area of the Golgi stack and the TGN of baby hamster kidney cells under different conditions. The results indicate that the increase in the size of the TGN at 20 degrees C is accompanied by a significant decrease in the surface area and volume of the preceding Golgi compartments. A similar effect is also seen in uninfected cells at 20 degrees C, as well as during normal (37 degrees C) infection with Semliki Forest virus. In the latter case, however, the decrease in the size of the Golgi stack and the increase in that of the TGN is not accompanied by inhibition of transport from the Golgi complex to the cell surface. The results indicate that the Golgi stack and the TGN are dynamic and interrelated structures that are capable of rapid alteration in total surface area in response to changes in the rates of membrane transport.     

The new face of active receptor bound arrestin attracts new partners

Conformational change in arrestin induced by receptor binding promotes its interaction with the majority of recently identified nonreceptor binding partners.     

Neuregulin-1 type III determines the ensheathment fate of axons

The signals that determine whether axons are ensheathed or myelinated by Schwann cells have long been elusive. We now report that threshold levels of neuregulin-1 (NRG1) type III on axons determine their ensheathment fate. Ensheathed axons express low levels whereas myelinated fibers express high levels of NRG1 type III. Sensory neurons from NRG1 type III deficient mice are poorly ensheathed and fail to myelinate; lentiviral-mediated expression of NRG1 type III rescues these defects. Expression also converts the normally unmyelinated axons of sympathetic neurons to myelination. Nerve fibers of mice haploinsufficient for NRG1 type III are disproportionately unmyelinated, aberrantly ensheathed, and hypomyelinated, with reduced conduction velocities. Type III is the sole NRG1 isoform retained at the axon surface and activates PI 3-kinase, which is required for Schwann cell myelination. These results indicate that levels of NRG1 type III, independent of axon diameter, provide a key instructive signal that determines the ensheathment fate of axons.     

Alternative splicing determines the post-endocytic sorting fate of G-protein-coupled receptors

Mu-type opioid receptors are physiologically important G-protein-coupled receptors that are generally thought to recycle after agonist-induced endocytosis. Here we show that several alternatively spliced receptor variants fail to do so efficiently because of splice-mediated removal of an endocytic sorting sequence that is present specifically in the MOR1 variant. All of the recycling-impaired receptor variants were found to undergo proteolytic down-regulation more rapidly than MOR1, irrespective of moderate differences in endocytic rate, indicating that alternative splicing plays a specific role in distinguishing the trafficking itinerary of receptors after endocytosis. The recycling-impaired MOR1B variant was similar to MOR1 in its ability to mediate opioid-dependent inhibition of adenylyl cyclase, and to undergo opioid-induced desensitization in intact cells. Functional recovery (resensitization) of MOR1B-mediated cellular responsiveness after opioid removal, however, was significantly impaired (4-fold reduction in rate) compared with MOR1. To our knowledge the present results are the first to establish a role of alternative RNA processing in specifying the post-endocytic sorting of G-protein-coupled receptors between divergent and functionally distinct membrane pathways.     

The fate of internalized alpha-2-macroglobulin: alpha-galactosidase conjugate in fibroblasts from Fabry's hemizygote

In our previous report we described the endocytotic incorporation of coffee bean alpha-galactosidase conjugated to human alpha-2-macroglobulin (alpha-2-M) into cultured fibroblasts derived from a patient with Fabry's disease (1). The fate of internalized alpha-galactosidase according to the method described in the above report is now studied. Measurement of the enzyme activity of subcellular fractions showed that it was concentrated in the lysosomal-mitochondrial fraction. The half-life of internalized alpha-galactosidase was determined to be 2 h.     

The dopamine D4 receptor,the ultimate disordered protein

The human D4 dopamine receptor is a synaptic neurotransmitter receptor responsible for neuronal signaling in the mesolimbic system of the brain, an area of the brain that regulates emotion and complex behavior. Its structure makes it a very unusual and interesting G protein-coupled receptor (GPCR) as it has several polymorphic variants of its gene in the region encoding the third intracellular loop (IL3). This region contains from two to seven or more similar 48 base pair repeats. These repeats cause this protein to have a very high disorder index and this, in turn, makes it very interactive with other proteins. Among GPCRs in general, the unusually proline-rich IL3 is unique to the D4 receptor (D4R). We believe that, as in the D2R, this region of the receptor plays a role in it’s interaction with other receptors.     

The differential engagement of arrestin surface charges by the various functional forms of the receptor

G-protein-coupled receptor signaling is terminated by arrestin proteins that preferentially bind to the activated phosphorylated form of the receptor. Arrestins also bind active unphosphorylated and inactive phosphorylated receptors. Binding to the non-preferred forms of the receptor is important for visual arrestin translocation in rod photoreceptors and the regulation of receptor signaling and trafficking by non-visual arrestins. Given the importance of arrestin interactions with the various functional forms of the receptor, we performed an extensive analysis of the receptor-binding surface of arrestin using site-directed mutagenesis. The data indicated that a large number of surface charges are important for arrestin interaction with all forms of the receptor. Arrestin elements involved in receptor binding are differentially engaged by the various functional forms of the receptor, each requiring a unique subset of arrestin residues in a specific spatial configuration. We identified several additional phosphate-binding elements in the N-domain and demonstrated for the first time that the active receptor preferentially engages the arrestin C-domain. We also found that the interdomain contact surface is important for arrestin interaction with the non-preferred forms of the receptor and that residues in this region play a role in arrestin transition into its high affinity receptor binding state.     

The complex nature of protein phosphatases

Protein phosphatases are integrally associated with the regulation of cellular signaling. The mechanisms underlying the specific regulatory roles are likely to be unique to each cell system. Nevertheless, analysis of phosphatase regulation in a number of systems has identified phosphatase targeting through association with a wide range of binding partners to be a fundamental mechanism of regulation. Using protein phosphatase 2A (PP2A) as an example, this snapshot summarizes these fundamental mechanisms of protein phosphatase regulation.     

G protein-coupled receptor/arrestin3 modulation of the endocytic machinery

Nonvisual arrestins (arr) modulate G protein-coupled receptor (GPCR) desensitization and internalization and bind to both clathrin (CL) and AP-2 components of the endocytic coated pit (CP). This raises the possibility that endocytosis of some GPCRs may be a consequence of arr-induced de novo CP formation. To directly test this hypothesis, we examined the behavior of green fluorescent protein (GFP)-arr3 in live cells expressing beta2-adrenergic receptors and fluorescent CL. After agonist stimulation, the diffuse GFP-arr3 signal rapidly became punctate and colocalized virtually completely with preexisting CP spots, demonstrating that activated complexes accumulate in previously formed CPs rather than nucleating new CP formation. After arr3 recruitment, CP appeared larger: electron microscopy analysis revealed an increase in both CP number and in the occurrence of clustered CPs. Mutant arr3 proteins with impaired binding to CL or AP-2 displayed reduced recruitment to CPs, but were still capable of inducing CP clustering. In contrast, though constitutively present in CPs, the COOH-terminal moiety of arr3, which contains CP binding sites but lacks receptor binding, did not induce CP clustering. Together, these results indicate that recruitment of functional arr3-GPCR complexes to CP is necessary to induce clustering. Latrunculin B or 16 degrees C blocked CP rearrangements without affecting arr3 recruitment to CP. These results and earlier studies suggest that discrete CP zones exist on cell surfaces, each capable of supporting adjacent CPs, and that the cortical actin membrane skeleton is intimately involved with both the maintenance of existing CPs and the generation of new structures.     

The beta subunit determines the ion selectivity of the GABAA receptor

The gamma-aminobutyric acid, type A (GABA(A)) receptor is a chloride-conducting receptor composed of alpha, beta, and gamma subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1-M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1-M2 loop of the beta(3) subunit to the corresponding amino acids of the alpha(7) nicotinic acetylcholine subunit rendered the GABA(A) receptor cation-selective upon co-expression with wild type alpha(2) and gamma(2) subunits. Similar mutations in the alpha(2) or gamma(2) subunits did not lead to such a change in ion selectivity. This suggests a unique role for the beta(3) subunit in determining the ion selectivity of the GABA(A) receptor. The pharmacology of the mutated GABA(A) receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn(2+) and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC(50) = 1.3 microm) compared with the wild type receptor (EC(50) = 6.4 microm) and a loss of desensitization to GABA of the mutant receptor.     

Routing of internalized ricin and ricin conjugates to the Golgi complex

Receptor-mediated endocytosis and intracellular routing of native ricin, and of ricin conjugated to colloidal gold (Ri-Au) and to horseradish peroxidase (Ri-HRP), have been studied in cultured MCF-7 and Vero cells by electron microscopical techniques including serial section analysis. Both native ricin, as demonstrated by immunoperoxidase cytochemistry, and the ricin conjugates were internalized via a common coated pit-coated vesicle pathway to reach vacuolar and tubulo-vesicular portions of the endosomal system. In addition, native ricin and a purified monovalent fraction of Ri-HRP reached distinct Golgi cisterns, whereas Ri-Au and polyvalent Ri-HRP did not. The results delineate intracellular routing of native ricin and compare it with the routing of different ricin conjugates. Moreover, our study shows that conjugates of a particular ligand (ricin) and various probes (e.g., gold and peroxidase), may be handled differently by cells. Sorting apparently takes place in the endosomal system, allowing some but not other molecules to reach Golgi elements. This sorting seems to depend on the valency of the ricin conjugate.