Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice.

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 showed marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation was unchanged. In contrast, the angiotensin II-induced rise in contractility was significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.

The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.

An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.

Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G beta gamma activation of p21ras.

Stimulation of Gi-coupled receptors leads to the activation of mitogen-activated protein kinases (MAP kinases). In several cell types, this appears to be dependent on the activation of p21ras (Ras). Which G-protein subunit(s) (G alpha or the G beta gamma complex) primarily is responsible for triggering this signaling pathway, however, is unclear. We have demonstrated previously that the carboxyl terminus of the beta-adrenergic receptor kinase, containing its G beta gamma-binding domain, is a cellular G beta gamma antagonist capable of specifically distinguishing G alpha- and G beta gamma-mediated processes. Using this G beta gamma inhibitor, we studied Ras and MAP kinase activation through endogenous Gi-coupled receptors in Rat-1 fibroblasts and through receptors expressed by transiently transfected COS-7 cells. We report here that both Ras and MAP kinase activation in response to lysophosphatidic acid is markedly attenuated in Rat-1 cells stably transfected with a plasmid encoding this G beta gamma antagonist. Likewise in COS-7 cells transfected with plasmids encoding Gi-coupled receptors (alpha 2-adrenergic and M2 muscarinic), the activation of Ras and MAP kinase was significantly reduced in the presence of the coexpressed G beta gamma antagonist. Ras-MAP kinase activation mediated through a Gq-coupled receptor (alpha 1-adrenergic) or the tyrosine kinase epidermal growth factor receptor was unaltered by this G beta gamma antagonist. These results identify G beta gamma as the primary mediator of Ras activation and subsequent signaling via MAP kinase in response to stimulation of Gi-coupled receptors.

Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48-90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48-65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09 ± 0.02 vs. 1.77 ± 0.01 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0 ± 1.1 mN and 11.8 ± 4.5 mN/mm(2), respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7 ± 1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7 ± 0.3-fold force increase, EC50 = 95.1 nm). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.

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.

Adjunctive albuterol enhances the response to enzyme replacement therapy in late-onset Pompe disease.

Effective dosages for enzyme replacement therapy (ERT) in Pompe disease are much higher than for other lysosomal storage disorders, which has been attributed to low cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle. We have previously demonstrated the benefit of increased CI-MPR-mediated uptake of recombinant human acid-α-glucosidase during ERT in mice with Pompe disease following addition of albuterol therapy. Currently we have completed a pilot study of albuterol in patients with late-onset Pompe disease already on ERT for >2 yr, who were not improving further. The 6-min walk test (6MWT) distance increased in all 7 subjects at wk 6 (30±13 m; P=0.002), wk 12 (34±14 m; P=0.004), and wk 24 (42±37 m; P=0.02), in comparison with baseline. Grip strength was improved significantly for both hands at wk 12. Furthermore, individual subjects reported benefits; e.g., a female patient could stand up from sitting on the floor much more easily (time for supine to standing position decreased from 30 to 11 s), and a male patient could readily swing his legs out of his van seat (hip abduction increased from 1 to 2+ on manual muscle testing). Finally, analysis of the quadriceps biopsies suggested increased CI-MPR at wk 12 (P=0.08), compared with baseline. With the exception of 1 patient who succumbed to respiratory complications of Pompe disease in the first week, only mild adverse events have been reported, including tremor, transient difficulty falling asleep, and mild urinary retention (requiring early morning voiding). Therefore, this pilot study revealed initial safety and efficacy in an open label study of adjunctive albuterol therapy in patients with late-onset Pompe disease who had been stable on ERT with no improvements noted over the previous several years.

Adjunctive β2-agonists reverse neuromuscular involvement in murine Pompe disease.

Pompe disease has resisted enzyme replacement therapy with acid α-glucosidase (GAA), which has been attributed to inefficient cation-independent mannose-6-phosphate receptor (CI-MPR) mediated uptake. We evaluated β2-agonist drugs, which increased CI-MPR expression in GAA knockout (KO) mice. Clenbuterol along with a low-dose adeno-associated virus vector increased Rotarod latency by 75% at 4 wk, in comparison with vector alone (P<2×10(-5)). Glycogen content was lower in skeletal muscles, including soleus (P<0.01), extensor digitorum longus (EDL; P<0.001), and tibialis anterior (P<0.05) following combination therapy, in comparison with vector alone. Glycogen remained elevated in the muscles following clenbuterol alone, indicating an adjunctive effect with gene therapy. Elderly GAA-KO mice treated with combination therapy demonstrated 2-fold increased wirehang latency, in comparison with vector or clenbuterol alone (P<0.001). The glycogen content of skeletal muscle decreased following combination therapy in elderly mice (P<0.05). Finally, CI-MPR-KO/GAA-KO mice did not respond to combination therapy, indicating that clenbuterol's effect depended on CI-MPR expression. In summary, adjunctive β2-agonist treatment increased CI-MPR expression and enhanced efficacy from gene therapy in Pompe disease, which has implications for other lysosomal storage disorders that involve primarily the brain.

Reliability of blood pressure determination with the Finapres with altered physiological states or pharmacodynamic conditions

The blood pressure waveform is modified on distal propagation by phenomena such as dispersion, reflection and the state of the arterial compliance. The consequent effects are amplification and narrowing of the wave, with an increased systolic, reduced diastolic and essentially unaltered mean blood pressure. The Finapres measures the peripheral pressure using the volume clamp principle; it has not been validated under altered physiological conditions and during pharmacodynamic interventions. We studied simultaneous Finapres and brachial blood pressures (using a conventional oscillometric sphygmomanometer—Vitalmap) in ten normal volunteers at rest, and during dynamic exercise and a cold pressor test. The effects of pharmacodynamic intervention were examined following beta-adrenoceptor blockade with propranolol (160 mg) or beta-adrenoceptor modulation with the beta-adrenoceptor partial agonist celiprolol (400 mg). The Finapres systolic pressure was significantly higher than the brachial value during all three test states. The difference between the systolic pressures measured by the two devices was shown to increase significantly during the cold pressor test, but not during dynamic (supine bicycle) exercise. The Finapres diastolic pressure was significantly higher than the Vitalmap value during exercise and the cold pressor test. The differences between the two methods increased significantly over time. Beta-adrenergic blockade with propranolol or modulation with celiprolol had no significant interaction with the pressure differences between the Finapres and Vitalmap techniques. The results would support the view that the Finapres can provide blood pressure information which is robust under most circumstances. Although this pharmacodynamic intervention did not alter the relationship between the peripheral and central blood pressure, it is important to note that this dynamic relationship is sensitive to circulatory loading conditions and wave transmission characteristics; it is possible that the Finapres could be less reliable in clinical settings where potent vasoactive agents were being administered.

Rhodopsin and the Others: A Historical Perspective on Structural Studies of G Protein-Coupled Receptors

The role of rhodopsin as a structural prototype for the study of the whole superfamily of G protein-coupled receptors (GPCRs) is reviewed in an historical perspective. Discovered at the end of the nineteenth century, fully sequenced since the early 1980s, and with direct three-dimensional information available since the 1990s, rhodopsin has served as a platform to gather indirect information on the structure of the other superfamily members. Recent breakthroughs have elicited the solution of the structures of additional receptors, namely the beta 1- and beta 2-adrenergic receptors and the A(2A) adenosine receptor, now providing an opportunity to gauge the accuracy of homology modeling and molecular docking techniques and to perfect the computational protocol. Notably, in coordination with the solution of the structure of the A(2A) adenosine receptor, the first "critical assessment of GPCR structural modeling and docking" has been organized, the results of which highlighted that the construction of accurate models, although challenging, is certainly achievable. The docking of the ligands and the scoring of the poses clearly emerged as the most difficult components. A further goal in the field is certainly to derive the structure of receptors in their signaling state, possibly in complex with agonists. These advances, coupled with the introduction of more sophisticated modeling algorithms and the increase in computer power, raise the expectation for a substantial boost of the robustness and accuracy of computer-aided drug discovery techniques in the coming years.

Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the beta-agonist

Ractopamine (RCT) is a phenethanolamine member of the family of beta-adrenergic agonists (beta-agonists), This class of compounds have become notable for their properties of enhancing the growth rates of farm animal species but are not licensed for use in Europe. An ELISA procedure employing a polyclonal antibody raised in a goat was developed to detect RCT residues in bovine urine samples, The assay had a high sensitivity (calibration curve mid-point of 22 pg per well), allowing the analysis of urine samples without the need for sample clean-up. In addition, an LC-MS-MS confirmatory procedure was developed which was able to act as a confirmatory procedure for the ELISA results. Four calves were orally treated with RCT (0.1 mg kg(-1) body mass for 17 d) and urine samples collected were assayed by both analytical procedures. It was observed that RCT residues were excreted mainly in the form of glucuronides and deconjugation could be achieved using two different sources of the enzyme beta-glucuronidase (Helix pomatia and Escherichia coli), High concentrations of RCT residues were found throughout the medication period (44-473 ng ml(-1); LC-MS-MS data) and remained present for several days following removal of the drug from the diet, RCT residues were no longer detectable 2 weeks after withdrawal, Good agreement (r(2) = 0.73) was achieved between the ELISA and LC-MS-MS results, especially when sample deconjugation was applied to the urine samples for both sets of analyses, The results show that an effective screening and confirmatory system was devised to detect RCT residues in urine samples taken during treatment and close to withdrawal, However, alternative matrices may have to be selected to allow the illegal use of the substance to be detected following prolonged withdrawal times.

Comparison of Dynamics of Extracellular Accesses to the β(1) and β(2) Adrenoceptors Binding Sites Uncovers the Potential of Kinetic Basis of Antagonist Selectivity.

From the molecular mechanism of antagonist unbinding in the ß(1) and ß(2) adrenoceptors investigated by steered molecular dynamics, we attempt to provide further possibilities of ligand subtype and subspecies selectivity. We have simulated unbinding of ß(1) -selective Esmolol and ß(2) -selective ICI-118551 from both receptors to the extracellular environment and found distinct molecular features of unbinding. By calculating work profiles, we show different preference in antagonist unbinding pathways between the receptors, in particular, perpendicular to the membrane pathway is favourable in the ß(1) adrenoceptor, whereas the lateral pathway involving helices 5, 6 and 7 is preferable in the ß(2) adrenoceptor. The estimated free energy change of unbinding based on the preferable pathway correlates with the experimental ligand selectivity. We then show that the non-conserved K347 (6.58) appears to facilitate in guiding Esmolol to the extracellular surface via hydrogen bonds in the ß(1) adrenoceptor. In contrast, hydrophobic and aromatic interactions dominate in driving ICI-118551 through the easiest pathway in the ß(2) adrenoceptor. We show how our study can stimulate design of selective antagonists and discuss other possible molecular reasons of ligand selectivity, involving sequential binding of agonists and glycosylation of the receptor extracellular surface. © 2012 John Wiley & Sons A/S.