Regions of beta 2 and beta 4 responsible for differences between the steady state dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 neuronal nicotinic receptors

We constructed chimeras of the rat beta 2 and beta 4 neuronal nicotinic subunits to locate the regions that contribute to differences between the acetylcholine (ACh) dose-response relationships of the alpha 3 beta 2 and alpha 3 beta 4 receptors. Expressed in Xenopus oocytes, the alpha 3 beta 2 receptor displays an EC50 for ACh approximately 20-fold less than the EC50 of the alpha 3 beta 4 receptor. The apparent Hill slope (n(app)) of alpha 3 beta 2 is near one whereas the alpha 3 beta 4 receptor displays an n(app) near two. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other. Exchanging just beta 2:104-120 for the corresponding region of beta 4 shifts the EC50 of ACh dose-response relationship in the expected direction but does not completely convert the EC50 of the dose- response relationship from one wild-type value to the other. However, substitutions in the beta 2:104-120 region do account for the relative sensitivity of the alpha 3 beta 2 receptor to cytisine, tetramethylammonium, and ACh. The expression of beta 4-like (strong) cooperativity requires an extensive region of beta 4 (beta 4:1-301). Relatively short beta 2 substitutions (beta 2:104-120) can reduce cooperativity to beta 2-like values. The results suggest that amino acids within the first 120 residues of beta 2 and the corresponding region of beta 4 contribute to an agonist binding site that bridges the alpha and beta subunits in neuronal nicotinic receptors

TGF beta type II receptor signaling controls Schwann cell death and proliferation in developing nerves

During development, Schwann cell numbers are precisely adjusted to match the number of axons. It is essentially unknown which growth factors or receptors carry out this important control in vivo. Here, we tested whether the type II transforming growth factor (TGF)beta receptor has a role in this process. We generated a conditional knock-out mouse in which the type II TGF beta receptor is specifically ablated only in Schwann cells. Inactivation of the receptor, evident at least from embryonic day 18, resulted in suppressed Schwann cell death in normally developing and injured nerves. Notably, the mutants also showed a strong reduction in Schwann cell proliferation. Consequently, Schwann cell numbers in wild-type and mutant nerves remained similar. Lack of TGF beta signaling did not appear to affect other processes in which TGF beta had been implicated previously, including myelination and response of adult nerves to injury. This is the first in vivo evidence for a growth factor receptor involved in promoting Schwann cell division during development and the first genetic evidence for a receptor that controls normal developmental Schwann cell death

Regulation of Membrane Targeting of the G Protein-coupled Receptor Kinase 2 by Protein Kinase A and Its Anchoring Protein AKAP79

The beta 2 adrenergic receptor (beta 2AR) undergoes desensitization by a process involving its phosphorylation by both protein kinase A (PKA) and G protein-coupled receptor kinases (GRKs). The protein kinase A-anchoring protein AKAP79 influences beta 2AR phosphorylation by complexing PKA with the receptor at the membrane. Here we show that AKAP79 also regulates the ability of GRK2 to phosphorylate agonist-occupied receptors. In human embryonic kidney 293 cells, overexpression of AKAP79 enhances agonist-induced phosphorylation of both the beta 2AR and a mutant of the receptor that cannot be phosphorylated by PKA (beta 2AR/PKA-). Mutants of AKAP79 that do not bind PKA or target to the beta 2AR markedly inhibit phosphorylation of beta 2AR/PKA-. We show that PKA directly phosphorylates GRK2 on serine 685. This modification increases Gbeta gamma subunit binding to GRK2 and thus enhances the ability of the kinase to translocate to the membrane and phosphorylate the receptor. Abrogation of the phosphorylation of serine 685 on GRK2 by mutagenesis (S685A) or by expression of a dominant negative AKAP79 mutant reduces GRK2-mediated translocation to beta 2AR and phosphorylation of agonist-occupied beta 2AR, thus reducing subsequent receptor internalization. Agonist-stimulated PKA-mediated phosphorylation of GRK2 may represent a mechanism for enhancing receptor phosphorylation and desensitization

Agrin-induced acetylcholine receptor clustering in mammalian muscle requires tyrosine phosphorylation.

Agrin is thought to be the nerve-derived factor that initiates acetylcholine receptor (AChR) clustering at the developing neuromuscularjunction. We have investigated the signaling pathway in mouse C2 myotubes and report that agrin induces a rapid but transient tyrosine phosphorylation of the AChR beta subunit. As the beta-subunit tyrosine phosphorylation occurs before the formation of AChR clusters, it may serve as a precursor step in the clustering mechanism. Consistent with this, we observed that tyrosine phosphorylation of the beta subunit correlated precisely with the presence or absence of clustering under several experimental conditions. Moreover, two tyrosine kinase inhibitors, herbimycin and staurosporine, that blocked beta-subunit phosphorylation also blocked agrin-induced clustering. Surprisingly, the inhibitors also dispersed preformed AChR clusters, suggesting that the tyrosine phosphorylation of other proteins may be required for the maintenance of receptor clusters. These findings indicate that in mammalian muscle, agrin-induced AChR clustering occurs through a mechanism that requires tyrosine phosphorylation and may involve tyrosine phosphorylation of the AChR itself

Regions of the T cell receptor alpha and beta chains that are responsible for interactions with CD3.

The T cell antigen receptor consists of the Ti alpha/beta heterodimer which recognizes antigen, and the associated CD3 chains, thought to be involved in signal transduction. To understand the nature of the interaction between Ti and CD3, chimeric molecules which included the COOH-terminal segments of Ti alpha or beta linked to the extracellular segment of CD8, were transfected into a mutant T cell deficient in Ti beta chain expression and cell surface CD3. Both chimeric chains were required to express the chimeric Ti and to restore CD3 surface expression. CD8/Ti and CD3 cointernalized and coimmunoprecipitated. Stimulation of the chimeric receptor induced transmembrane signaling events and cell activation. These results demonstrate that the Ti alpha and beta COOH termini containing the transmembrane domains are sufficient for structural and functional coupling of Ti to CD3

Beta-Adrenergic Receptor Blockade By Propranolol Enhances Retention In A Multitrial Passive-Avoidance Procedure

The effect of beta -adrenergic receptor blockade on retention in a mildly aversive passive-avoidance procedure was investigated. Rats were given passive-avoidance training-1 trial per day for 4 days-and were administered saline, the centrally and peripherally acting beta -adrenergic blocker propranolol (4 or 10 mg/kg ip), or the peripherally acting P-adrenergic blocker sotalol (4 or 10 mg/kg ip) immediately or 2 hr after the Ist trial. Enhanced retention occurred only with the higher dose (10 mg/kg) of propranolol and only when it was administered immediately after training. The enhanced retention produced by propranolol is discussed in terms of opposing, regionally specific actions of beta -adrenergic receptor-mediated neural circuits on modulation of memory

Endothelial LRP1 transports amyloid-β1-42 across the blood-brain barrier

According to the neurovascular hypothesis, impairment of low-density lipoprotein receptor-related protein-1 (LRP1) in brain capillaries of the blood-brain barrier (BBB) contributes to neurotoxic amyloid-beta (A beta) brain accumulation and drives Alzheimer's disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in A beta transport and the expression of LRP1 in brain endothelium, the role of LRP1 at the BBB is uncertain. As global Lrp1 deletion in mice is lethal, appropriate models to study the function of LRP1 are lacking. Moreover, the relevance of systemic A beta clearance to AD pathology remains unclear, as no BBB-specific knockout models have been available. Here, we developed transgenic mouse strains that allow for tamoxifen-inducible deletion of Lrp1 specifically within brain endothelial cells (Slo1c1-CreER(Tz) Lrp1(fl/fl) mice) and used these mice to accurately evaluate LRP1-mediated A beta BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [I-125] A beta(1-42). Additionally, in the 5xFAD mouse model of AD, brain endothelial-specific Lrp1 deletion reduced plasma A beta levels and elevated soluble brain A beta, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic AD elimination via the BBB. Together, our results suggest that receptor-mediated A beta BBB clearance may be a potential target for treatment and prevention of A beta brain accumulation in AD

Duality of β-glucan microparticles: antigen carrier and immunostimulants

Designing efficient recombinant mucosal vaccines against enteric diseases is still a major challenge. Mucosal delivery of recombinant vaccines requires encapsulation in potent immunostimulatory particles to induce an efficient immune response. This paper evaluates the capacity of beta-glucan microparticles (GPs) as antigen vehicles and characterizes their immune-stimulatory effects. The relevant infectious antigen FedF was chosen to be loaded inside the microparticles. The incorporation of FedF inside the particles was highly efficient (roughly 85%) and occurred without antigen degradation. In addition, these GPs have immunostimulatory effects as well, demonstrated by the strong reactive oxygen species (ROS) production by porcine neutrophils upon their recognition. Although antigen-loaded GPs still induce ROS production, antigen loading decreases this production by neutrophils for reasons yet unknown. However, these antigen-loaded GPs are still able to bind their specific beta-glucan receptor, demonstrated by blocking complement receptor 3, which is the major beta-glucan receptor on porcine neutrophils. The dual character of these particles is confirmed by a T-cell proliferation assay. FedF-loaded particles induce a significantly higher FedF-specific T-cell proliferation than soluble FedF. Taken together, these results show that GPs are efficient antigen carriers with immune-stimulatory properties