Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution.

Prolonged exposure of cells or tissues to drugs or hormones such as catecholamines leads to a state of refractoriness to further stimulation by that agent, known as homologous desensitization. In the case of the beta-adrenergic receptor coupled to adenylate cyclase, this process has been shown to be intimately associated with the sequestration of the receptors from the cell surface through a cAMP-independent process. Recently, we have shown that homologous desensitization in the frog erythrocyte model system is also associated with increased phosphorylation of the beta-adrenergic receptor. We now provide evidence that the phosphorylation state of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase, subcellular translocation, and recycling to the cell surface during the process of agonist-induced homologous desensitization. Moreover, we show that the receptor phosphorylation is reversed by a phosphatase specifically associated with the sequestered subcellular compartment. At 23 degrees C, the time courses of beta-adrenergic receptor phosphorylation, sequestration, and adenylate cyclase desensitization are identical, occurring without a lag, exhibiting a t1/2 of 30 min, and reaching a maximum at approximately 3 hr. Upon cell lysis, the sequestered beta-adrenergic receptors can be partially recovered in a light membrane vesicle fraction that is separable from the plasma membranes by differential centrifugation. The increased beta-adrenergic receptor phosphorylation is apparently reversed in the sequestered vesicle fraction as the sequestered receptors exhibit a phosphate/receptor stoichiometry that is similar to that observed under basal conditions. High levels of a beta-adrenergic receptor phosphatase activity appear to be associated with the sequestered vesicle membranes. The functional activity of the phosphorylated beta-adrenergic receptor was examined by reconstituting purified receptor with its biochemical effector the guanine nucleotide regulatory protein (Ns) in phospholipid vesicles and assessing the receptor-stimulated GTPase activity of Ns. Compared to controls, phosphorylated beta-adrenergic receptors, purified from desensitized cells, were less efficacious in activating the Ns GTPase activity. These results suggest that phosphorylation of the beta-adrenergic receptor leads to its functional uncoupling and physical translocation away from the cell surface into a sequestered membrane domain. In the sequestered compartment, the phosphorylation is reversed thus enabling the receptor to recycle back to the cell surface and recouple with adenylate cyclase.

Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors.

beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta-AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist-promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors.

Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor.

Agonist-promoted desensitization of adenylate cyclase is intimately associated with phosphorylation of the beta-adrenergic receptor in mammalian, avian, and amphibian cells. However, the nature of the protein kinase(s) involved in receptor phosphorylation remains largely unknown. We report here the identification and partial purification of a protein kinase capable of phosphorylating the agonist-occupied form of the purified beta-adrenergic receptor. The enzyme is prepared from a supernatant fraction from high-speed centrifugation of lysed kin- cells, a mutant of S49 lymphoma cells that lacks a functional cAMP-dependent protein kinase. The beta-agonist isoproterenol induces a 5- to 10-fold increase in receptor phosphorylation by this kinase, which is blocked by the antagonist alprenolol. Fractionation of the kin- supernatant on molecular-sieve HPLC and DEAE-Sephacel results in a 50- to 100-fold purified beta-adrenergic receptor kinase preparation that is largely devoid of other protein kinase activities. The kinase activity is insensitive to cAMP, cGMP, cAMP-dependent kinase inhibitor, Ca2+-calmodulin, Ca2+-phospholipid, and phorbol esters and does not phosphorylate general kinase substrates such as casein and histones. Phosphate appears to be incorporated solely into serine residues. The existence of this novel cAMP-independent kinase, which preferentially phosphorylates the agonist-occupied form of the beta-adrenergic receptor, suggests a mechanism that may explain the homologous or agonist-specific form of adenylate cyclase desensitization. It also suggests a general mechanism for regulation of receptor function in which only the agonist-occupied or "active" form of the receptor is a substrate for enzymes inducing covalent modification.

Phorbol esters promote alpha 1-adrenergic receptor phosphorylation and receptor uncoupling from inositol phospholipid metabolism.

DDT1 MF-2 cells, which are derived from hamster vas deferens smooth muscle, contain alpha 1-adrenergic receptors (54,800 +/- 2700 sites per cell) that are coupled to stimulation of inositol phospholipid metabolism. Incubation of these cells with tumor-promoting phorbol esters, which stimulate calcium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of alpha 1-receptor agonists such as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32P content of phosphatidylinositol and phosphatidic acid after prelabeling of the cellular ATP pool with 32Pi. These phorbol ester-treated cells also displayed a decrease in binding affinity of cellular alpha 1 receptors for agonists with no change in antagonist affinity. By using affinity chromatography on the affinity resin Affi-Gel-A55414, the alpha 1 receptors were purified approximately equal to 300-fold from control and phorbol ester-treated 32Pi-prelabeled cells. As assessed by NaDodSO4/polyacrylamide gel electrophoresis, the Mr 80,000 alpha 1-receptor ligand-binding subunit is a phosphopeptide containing 1.2 mol of phosphate per mol of alpha 1 receptor. After phorbol ester treatment this increased to 3.6 mol of phosphate per mol of alpha 1 receptor. The effect of phorbol esters on norepinephrine-stimulated inositol phospholipid turnover and alpha 1-receptor phosphorylation showed the same rapid time course with a t1/2 less than 2 min. These results indicate that calcium- and phospholipid-dependent protein kinase may play an important role in regulating the function of receptors that are coupled to the inositol phospholipid cycle by phosphorylating and deactivating them.

Impaired formation of beta-adrenergic receptor-nucleotide regulatory protein complexes in pseudohypoparathyroidism.

Decreased activity of the guanine nucleotide regulatory protein (N) of the adenylate cyclase system is present in cell membranes of some patients with pseudohypoparathyrodism (PHP-Ia) whereas others have normal activity of N (PHP-Ib). Low N activity in PHP-Ia results in a decrease in hormone (H)-stimulatable adenylate cyclase in various tissues, which might be due to decreased ability to form an agonist-specific high affinity complex composed of H, receptor (R), and N. To test this hypothesis, we compared beta-adrenergic agonist-specific binding properties in erythrocyte membranes from five patients with PHP-Ia (N = 45% of control), five patients with PHP-Ib (N = 97%), and five control subjects. Competition curves that were generated by increasing concentrations of the beta-agonist isoproterenol competing with [125I]pindolol were shallow (slope factors less than 1) and were computer fit to a two-state model with corresponding high and low affinity for the agonist. The agonist competition curves from the PHP-Ia patients were shifted significantly (P less than 0.02) to the right as a result of a significant (P less than 0.01) decrease in the percent of beta-adrenergic receptors in the high affinity state from 64 +/- 22% in PHP-Ib and 56 +/- 5% in controls to 10 +/- 8% in PHP-Ia. The agonist competition curves were computer fit to a "ternary complex" model for the two-step reaction: H + R + N in equilibrium HR + N in equilibrium HRN. The modeling was consistent with a 60% decrease in the functional concentration of N, and was in good agreement with the biochemically determined decrease in erythrocyte N protein activity. These in vitro findings in erythrocytes taken together with the recent observations that in vivo isoproterenol-stimulated adenylate cyclase activity is decreased in patients with PHP (Carlson, H. E., and A. S. Brickman, 1983, J. Clin. Endocrinol. Metab. 56:1323-1326) are consistent with the notion that N is a bifunctional protein interacting with both R and the adenylate cyclase. It may be that in patients with PHP-Ia a single molecular and genetic defect accounts for both decreased HRN formation and decreased adenylate cyclase activity, whereas in PHP-Ib the biochemical lesion(s) appear not to affect HRN complex formation.

Changes in midbrain pain receptor expression, gait and behavioral sensitivity in a rat model of radiculopathy.

Intervertebral disc herniation may contribute to inflammatory processes that associate with radicular pain and motor deficits. Molecular changes at the affected dorsal root ganglion (DRG), spinal cord, and even midbrain, have been documented in rat models of radiculopathy or nerve injury. The objective of this study was to evaluate gait and the expression of key pain receptors in the midbrain in a rodent model of radiculopathy. Radiculopathy was induced by harvesting tail nucleus pulposus (NP) and placing upon the right L5 DRG in rats (NP-treated, n=12). Tail NP was discarded in sham-operated animals (n=12). Mechanical allodynia, weight-bearing, and gait were evaluated in all animals over time. At 1 and 4 weeks after surgery, astrocyte and microglial activation was tested in DRG sections. Midbrain sections were similarly evaluated for immunoreactivity to serotonin (5HT(2B)), mu-opioid (µ-OR), and metabotropic glutamate (mGluR4 and 5) receptor antibodies. NP-treated animals placed less weight on the affected limb 1 week after surgery and experienced mechanical hypersensitivity over the duration of the study. Astroctye activation was observed at DRGs only at 4 weeks after surgery. Findings for pain receptors in the midbrain of NP-treated rats included an increased expression of 5HT(2B) at 1, but not 4 weeks; increased expression of µ-OR and mGluR5 at 1 and 4 weeks (periaqueductal gray region only); and no changes in expression of mGluR4 at any point in this study. These observations provide support for the hypothesis that the midbrain responds to DRG injury with a transient change in receptors regulating pain responses.

The missense of smell: functional variability in the human odorant receptor repertoire.

Humans have ~400 intact odorant receptors, but each individual has a unique set of genetic variations that lead to variation in olfactory perception. We used a heterologous assay to determine how often genetic polymorphisms in odorant receptors alter receptor function. We identified agonists for 18 odorant receptors and found that 63% of the odorant receptors we examined had polymorphisms that altered in vitro function. On average, two individuals have functional differences at over 30% of their odorant receptor alleles. To show that these in vitro results are relevant to olfactory perception, we verified that variations in OR10G4 genotype explain over 15% of the observed variation in perceived intensity and over 10% of the observed variation in perceived valence for the high-affinity in vitro agonist guaiacol but do not explain phenotype variation for the lower-affinity agonists vanillin and ethyl vanillin.

An immunoglobulin C kappa-reactive single chain antibody fusion protein induces tolerance through receptor editing in a normal polyclonal immune system.

Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tolerance primarily or exclusively by receptor editing. We also demonstrate the unique advantages of our model in the genetic and cellular analysis of immune tolerance.

UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity.

The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extracellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40-binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.

Irisin and FNDC5 in retrospect: An exercise hormone or a transmembrane receptor?

FNDC5 (fibronectin domain-containing [protein] 5) was initially discovered and characterized by two groups in 2002. In 2011 FNDC5 burst into prominence as the parent of irisin, a small protein containing the fibronectin type III domain. Irisin was proposed to be secreted by skeletal muscle cells in response to exercise, and to circulate to fat tissue where it induced a transition to brown fat. Since brown fat results in dissipation of energy, this pathway is of considerable interest for metabolism and obesity. Here I review the original discoveries of FNDC5 and the more recent discovery of irisin. I note in particular three problems in the characterization of irisin: the antibodies used to detect irisin in plasma lack validity; the recombinant protein used to demonstrate activity in cell culture was severely truncated; and the degree of shedding of soluble irisin from the cell surface has not been quantitated. The original discovery proposing that FNDC5 may be a transmembrane receptor may deserve a new look.

Interleukin-15 receptor blockade in non-human primate kidney transplantation.

BACKGROUND: Interleukin (IL)-15 is a chemotactic factor to T cells. It induces proliferation and promotes survival of activated T cells. IL-15 receptor blockade in mouse cardiac and islet allotransplant models has led to long-term engraftment and a regulatory T-cell environment. This study investigated the efficacy of IL-15 receptor blockade using Mut-IL-15/Fc in an outbred non-human primate model of renal allotransplantation. METHODS: Male cynomolgus macaque donor-recipient pairs were selected based on ABO typing, major histocompatibility complex class I typing, and carboxy-fluorescein diacetate succinimidyl ester-based mixed lymphocyte responses. Once animals were assigned to one of six treatment groups, they underwent renal transplantation and bilateral native nephrectomy. Serum creatinine level was monitored twice weekly and as indicated, and protocol biopsies were performed. Rejection was defined as a increase in serum creatinine to 1.5 mg/dL or higher and was confirmed histologically. Complete blood counts and flow cytometric analyses were performed periodically posttransplant; pharmacokinetic parameters of Mut-IL-15/Fc were assessed. RESULTS: Compared with control animals, Mut-IL-15/Fc-treated animals did not demonstrate increased graft survival despite adequate serum levels of Mut-IL-15/Fc. Flow cytometric analysis of white blood cell subgroups demonstrated a decrease in CD8 T-cell and natural killer cell numbers, although this did not reach statistical significance. Interestingly, two animals receiving Mut-IL-15/Fc developed infectious complications, but no infection was seen in control animals. Renal pathology varied widely. CONCLUSIONS: Peritransplant IL-15 receptor blockade does not prolong allograft survival in non-human primate renal transplantation; however, it reduces the number of CD8 T cells and natural killer cells in the peripheral blood.

Exhausted CD8 T cells downregulate the IL-18 receptor and become unresponsive to inflammatory cytokines and bacterial co-infections.

During many chronic infections virus-specific CD8 T cells succumb to exhaustion as they lose their ability to respond to antigenic activation. Combinations of IL-12, IL-18, and IL-21 have been shown to induce the antigen-independent production of interferon (IFN)-γ by effector and memory CD8 T cells. In this study we investigated whether exhausted CD8 T cells are sensitive to activation by these cytokines. We show that effector and memory, but not exhausted, CD8 T cells produce IFN-γ and upregulate CD25 following exposure to certain combinations of IL-12, IL-18, and IL-21. The unresponsiveness of exhausted CD8 T cells is associated with downregulation of the IL-18-receptor-α (IL-18Rα). Although IL-18Rα expression is connected with the ability of memory CD8 T cells to self-renew and efflux rhodamine 123, the IL-18Rα(lo) exhausted cells remained capable of secreting this dye. To further evaluate the consequences of IL-18Rα downregulation, we tracked the fate of IL-18Rα-deficient CD8 T cells in chronically infected mixed bone marrow chimeras and discovered that IL-18Rα affects the initial but not later phases of the response. The antigen-independent responsiveness of exhausted CD8 T cells was also investigated following co-infection with Listeria monocytogenes, which induces the expression of IL-12 and IL-18. Although IL-18Rα(hi) memory cells upregulated CD25 and produced IFN-γ, the IL-18Rα(lo) exhausted cells failed to respond. Collectively, these findings indicate that as exhausted T cells adjust to the chronically infected environment, they lose their susceptibility to antigen-independent activation by cytokines, which compromises their ability to detect bacterial co-infections.

Adjunctive β2-agonist treatment reduces glycogen independently of receptor-mediated acid α-glucosidase uptake in the limb muscles of mice with Pompe disease.

Enzyme or gene replacement therapy with acid α-glucosidase (GAA) has achieved only partial efficacy in Pompe disease. We evaluated the effect of adjunctive clenbuterol treatment on cation-independent mannose-6-phosphate receptor (CI-MPR)-mediated uptake and intracellular trafficking of GAA during muscle-specific GAA expression with an adeno-associated virus (AAV) vector in GAA-knockout (KO) mice. Clenbuterol, which increases expression of CI-MPR in muscle, was administered with the AAV vector. This combination therapy increased latency during rotarod and wirehang testing at 12 wk, in comparison with vector alone. The mean urinary glucose tetrasaccharide (Glc4), a urinary biomarker, was lower in GAA-KO mice following combination therapy, compared with vector alone. Similarly, glycogen content was lower in cardiac and skeletal muscle following 12 wk of combination therapy in heart, quadriceps, diaphragm, and soleus, compared with vector alone. These data suggested that clenbuterol treatment enhanced trafficking of GAA to lysosomes, given that GAA was expressed within myofibers. The integral role of CI-MPR was demonstrated by the lack of effectiveness from clenbuterol in GAA-KO mice that lacked CI-MPR in muscle, where it failed to reverse the high glycogen content of the heart and diaphragm or impaired wirehang performance. However, the glycogen content of skeletal muscle was reduced by the addition of clenbuterol in the absence of CI-MPR, as was lysosomal vacuolation, which correlated with increased AKT signaling. In summary, β2-agonist treatment enhanced CI-MPR-mediated uptake and trafficking of GAA in mice with Pompe disease, and a similarly enhanced benefit might be expected in other lysosomal storage disorders.

Olfactory Receptor Subgenomes Linked with Broad Ecological Adaptations in Sauropsida.

Olfactory receptors (ORs) govern a prime sensory function. Extant birds have distinct olfactory abilities, but the molecular mechanisms underlining diversification and specialization remain mostly unknown. We explored OR diversity in 48 phylogenetic and ecologically diverse birds and 2 reptiles (alligator and green sea turtle). OR subgenomes showed species- and lineage-specific variation related with ecological requirements. Overall 1,953 OR genes were identified in reptiles and 16,503 in birds. The two reptiles had larger OR gene repertoires (989 and 964 genes, respectively) than birds (182-688 genes). Overall, birds had more pseudogenes (7,855) than intact genes (1,944). The alligator had significantly more functional genes than sea turtle, likely because of distinct foraging habits. We found rapid species-specific expansion and positive selection in OR14 (detects hydrophobic compounds) in birds and in OR51 and OR52 (detect hydrophilic compounds) in sea turtle, suggestive of terrestrial and aquatic adaptations, respectively. Ecological partitioning among birds of prey, water birds, land birds, and vocal learners showed that diverse ecological factors determined olfactory ability and influenced corresponding olfactory-receptor subgenome. OR5/8/9 was expanded in predatory birds and alligator, suggesting adaptive specialization for carnivory. OR families 2/13, 51, and 52 were correlated with aquatic adaptations (water birds), OR families 6 and 10 were more pronounced in vocal-learning birds, whereas most specialized land birds had an expanded OR family 14. Olfactory bulb ratio (OBR) and OR gene repertoire were correlated. Birds that forage for prey (carnivores/piscivores) had relatively complex OBR and OR gene repertoires compared with modern birds, including passerines, perhaps due to highly developed cognitive capacities facilitating foraging innovations.