Multiple interactions with many different partners are responsible for the amazing functional versatility of proteins, especially those participating in cellular regulation. The structural properties that could facilitate multiple interactions are examined for small GTPases. The role of cellular constraints, compartmentation and scaffolds on protein-protein interactions is considered.

We describe the synthesis of bordifluoropyrromethene (BODIPY), fluorescein, and related fluorescent derivatives of the beta-adrenergic ligand CGP 12177. With these probes we screened insect (Sf9) cells stably transformed with the human beta 2-adrenoceptor gene and expressing (2-3.5) x 10(5) human beta 2-adrenoceptors per cell. Among these derivatives only BODIPY-CGP gave a receptor-specific signal sufficiently strong for measuring the on- and off-rate constants and the equilibrium dissociation constant of beta-adrenoceptor-specific binding by spectrofluorometry or photon counting. Similar KD values for BODIPY-CGP binding were obtained by kinetic measurements (approx. 250 pM) and under equilibrium conditions (400 +/- 180 pM), and these were in the same range as those obtained with [3H]CGP 12177 (200 +/- 32 pM). The cell-bound fluorescence could be quenched specifically with nonfluorescent CGP 12177 to near background levels. The disposition of the beta 2-adrenoceptors in BODIPY-CGP-stained Sf9 cells was mainly restricted to the cell surface at 4 and 30 degrees C. Hence, beta-adrenoceptor-expressing cells can be stained specifically with BODIPY-CGP, and beta-adrenoceptors on a single cell can be assessed by photon counting under the fluorescence microscope. Cells can also be scanned by fluorescence-activated flow cytometry.

Protein ScienceVolume 3, Issue 3 p. 528-532 RecollectionFree Access Excursions in biophysics by a classical enzymologist Ernst J.M. Helmreich, Corresponding Author Ernst J.M. Helmreich Medical Clinic, Division of Clinical Biochemistry and Pathobiochemistry, The University of Wiirzburg School of Medicine, 97078 Würzburg, Federal Republic of GermanyMedical Clinic, Division of Clinical Biochemistry and Pathobiochemistry, The University of Würzburg School of Medicine, Versbacher Str. 5, 97078 Würzburg, Federal Republic of GermanySearch for more papers by this author Ernst J.M. Helmreich, Corresponding Author Ernst J.M. Helmreich Medical Clinic, Division of Clinical Biochemistry and Pathobiochemistry, The University of Wiirzburg School of Medicine, 97078 Würzburg, Federal Republic of GermanyMedical Clinic, Division of Clinical Biochemistry and Pathobiochemistry, The University of Würzburg School of Medicine, Versbacher Str. 5, 97078 Würzburg, Federal Republic of GermanySearch for more papers by this author First published: March 1994 https://doi.org/10.1002/pro.5560030319Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. 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The effect of temperature on the allosteric transitions of rabbit skeletal muscle phosphorylase b. Biochemistry 7: 4543– 4556. Kirschner K, Eigen M, Bittman R, Vogt B. 1966. The binding of nicotin-amide-adenine dinucleotide to yeast D-glyceraldehyde-3-phosphate dehydrogenase: Temperature-jump relaxation studies on the mechanism of an allosteric enzyme. Proc Natl Acad Sci USA 56: 1661– 1667. Klein HW, Im MJ, Palm D, Helmreich EJM. 1984. Does pyridoxal 5′-phosphate function in glycogen phosphorylase as an electrophilic or a general acid catalyst. Biochemistry 23: 5853– 5861. Klein HW, Palm D, Helmreich EJM. 1982. General acid-base catalysis of α-glucan phosphorylases: Stereospecific glucosyl transfer from D-glucal is a pyridoxal 5′-phosphate and orthophosphate (arsenate) dependent reaction. Biochemistry 21: 6675– 6684. Kurstjens NP, Frohlich M, Dees C, Cantrill RC, Hekman M, Helmreich EJM. 1991. Binding of α- and βγ-subunits of Go to β1-adrenoceptor in sealed unilamellar lipid vesicles. 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A mechanism for the phosphorylase reaction is proposed which offers a plausible explanation for the essential role of pyridoxal 5'-phosphate in glycogen phosphorylases: in the forward direction, phosphorolysis of alpha-1,4-glycosidic bonds in oligo- or polysaccharides is started by protonation of the glycosidic oxygen by the substrate orthophosphate followed by stabilization of the incipient oxocarbonium ion and subsequent covalent binding to form alpha-glucose 1-phosphate. In the reverse direction, protonation of the phosphate of glucose 1-phosphate destabilizes the glycosidic bond and promotes formation of a glucosyl oxocarbonium ion-phosphate anion pair. In the subsequent step the phosphate anion facilitates the nucleophilic attack of a terminal glucosyl residue on the carbonium ion bringing about alpha-1,4-glycosidic bond formation and primer elongation. Both in the forward and reverse reactions, the phosphate of the cofactor pyridoxal 5'-phosphate acts as a general acid (PL-OPO3H- or PL-OPO32-) and protonates the substrate phosphate functioning as proton shuttle. Thus in glycogen phosphorylases, phosphates which directly interact with each other have replaced a pair of amino acid carboxyl groups functioning in catalysis of carbohydrases.

Signal transduction by G-protein-coupled receptors is regulated by various mechanisms acting at the receptor level; those studied most thoroughly are from the beta-adrenergic receptor/Gs/adenylyl cyclase system. We report here a regulatory mechanism occurring at the level of the G proteins themselves. A protein with M(r) 33,000 that inhibits Gs-GTPase activity was purified from bovine brain. This protein is very similar or identical to phosducin, a protein previously thought to be specific for retina and pineal gland. Recombinant phosducin inhibited the GTPase activity of several G proteins, and also inhibited Gs-mediated adenylyl cyclase activation. Blockade of its inhibitory effects by protein kinase A suggests that phosducin may be part of a complex regulatory network controlling G-protein-mediated signalling.

Recent information obtained, mainly by recombinant cDNA technology, on structural heterogeneity of hormone and transmitter receptors, of GTP-binding proteins (G-proteins) and, especially, of G-protein-linked receptors is reviewed and the implications of structural heterogeneity for diversity of hormone and transmitter actions is discussed. For the future, three-dimensional structural analysis of membrane proteins participating in signal transmission and transduction pathways is needed in order to understand the molecular basis of allosteric regulatory mechanisms governing the interactions between these proteins including hysteretic properties and cell-cybernetic aspects.

A human cDNA fragment bearing the complete coding region for the beta 2-adrenergic receptor was introduced into the genome of Autographa california nuclear polyhedrosis virus under the control of the polyhedrin promoter. Binding studies using [125I]iodocyanopindolol showed that Sf9 insect cells infected with the recombinant virus expressed approximately 1 x 10(6) beta 2-adrenergic receptors on their cell surface. Photoaffinity labeling of whole cells and membranes revealed a molecular weight of approximately 46,000 for the expressed receptor. The receptor produced in insect cells is glycosylated but the extent and pattern differ from that of the receptor from human tissue. The heterologously expressed receptor was purified by alprenolol affinity chromatography, and was able to activate isolated Gs-protein.

The role of the βγ-subunits in the interaction of G-proteins was examined with β1-adrenoceptors purified from turkey erythrocytes and pure βγ-subunits prepared from turkey erythrocytes and bovine brain. On a non-denaturing polyacrylamide gel, the mobility of βγ-subunits was increased when incubated with β1-adrenoceptor and the β1-adrenergic agonist l-(−)-isoproterenol, whereas on incubation with the antagonist l-alprenolol the mobility was unchanged. Furthermore, the β1-adrenoceptor was retarded on a Sephadex G-50 column equilibrated with βγ-subunits and agonist. No retardation occurred in the presence of antagonist. These data suggest a direct interaction of activated β1-adrenoceptors with isolated βγ-subunits of G-proteins.

The synthesis and properties of a fluorescent derivative of the hydrophilic beta-adrenergic antagonist CGP-12177 are described. The fluorescence of the NBD derivative of CGP-12177 (CGP-NBD) is extremely sensitive to its environment, the quantum yield increasing 23-fold upon transfer from water to acetonitrile. This property of CGP-NBD was taken into account and a procedure was developed using quantitative chloroform extraction of ligand for the measurement of CGP-NBD bound specifically to beta-receptors on A431.E3 membranes. The fluorescent NBD-derivative of CGP-12177 bound strongly and specifically to A431 cells, a KD of 3.9 x 10(-10) M being measured; the specific binding represented 63% of the total binding at a concentration of 1 x 10(-8) M (256 x KD). A431.E3 cells were used for the binding studies since they gave consistently higher receptor numbers when compared with the native strain. A maximal number of 47,000 sites/cell and a KD of 100 pM were measured with CGP-12177 on adhered cells. The receptor number was strongly dependent upon cell density with only 3000 sites/cell being measured in suspension at confluence.

A431 cells incubated with tunicamycin (0.15 micrograms/ml) for 40 h under conditions where incorporation of [3H] leucine into protein was inhibited less than 10% expressed mainly a beta-receptor species of about Mr 40,000 which was ascribed to the nonglycosylated form of the beta-receptor of about Mr 75,000 found in normal A431 cells by photoaffinity labeling. However, the tunicamycin-treated cells expressed the same number of specific beta 2-receptor-binding sites as untreated cells. Moreover, the aglycoreceptors had the same ligand binding properties as beta-adrenoceptors from control cells; but, functional tests of the receptor from tunicamycin-treated cells in reconstituted lipid vesicles showed that receptors from tunicamycin-treated cells had lost coupling efficiency. The coupling defect was at the receptor level since control experiments indicated that the other components of the signal transmission chain from beta-adrenoceptor to adenylate cyclase, the stimulatory regulatory GTP-binding protein of adenylate cyclase and adenylate cyclase, were fully functional. Homologous desensitization in tunicamycin-treated cells was characterized by export from the cell surface and sequestration of about the same number of beta-adrenoceptors as in normal desensitized cells but without further reduction of hormonally stimulated adenylate cyclase below the low level already attained in nondesensitized tunicamycin-treated cells. This was explained by assuming that the receptors removed in the course of homologous desensitization from the surface of tunicamycin-treated cells were already nonfunctional. Thus, beta-adrenergic desensitization in tunicamycin-treated cells is characterized by the functional disengagement of receptor removal and loss of adenylate cyclase activity.

New fluorescent glucagon derivatives were synthesized by converting tryptophan 25 to 2-thiol-tryptophan with the consequent use of thiol specific fluorescent reagents. All derivatives retained the ability to bind tightly to rat liver membranes and rat hepatocytes in primary culture and to activate adenylate cyclase as potently as native glucagon. Thus these derivatives are full agonists. From experiments with monolayer cultured hepatocytes and 125 I-glucagon at elevated temperatures it was assumed that the ligand was internalised at this temperature since some of the specifically bound ligand could no longer be washed off with acid. This was confirmed in experiments where monolayer cultures of hepatocytes were incubated with the fluorescein-labelled derivates of glucagon, thus allowing the study of the distribution of glucagon specifically bound on the cell surface using video intensification microscopic techniques. In keeping with autoradiographic studies using radiolabelled glucagon, or electron microscope studies using ferritin labelled glucagon, we could now show using fluorescently labelled glucagon derivatives and video intensification microscopy that at lower temperatures the bound ligand was distributed all over the cell surface. Whereas, at the higher temperature, ligand derived fluorescence could only be detected in mobile intracellular vesicles following internalisation and removal from the cell surface.

Both mechanical loading and autophagy play important roles in regulating bone growth and remodeling, but the relationship between the two remains unclear. In this study, we examined bone structure with micro-CT imaging and measured bone mechanical properties with three-point bending experiments using bones from wild-type (WT) mice and conditional knockout (cKO) mice with Atg7 deletion in their osteoblasts. We found that the knockout mice had significantly less bone volume, bone thickness, bone ultimate breaking force, and bone stiffness compared to wild-type mice. Additionally, bone marrow cells from knockout mice had reduced differentiation and mineralization capacities in terms of alkaline phosphatase and calcium secretion, as well as Runx2 and osteopontin expression. Knockout mice also had significantly less relative bone formation rate due to mechanical loading. Furthermore, we found that the osteoblasts from wild-type mice had stronger responses to mechanical stimulation compared to autophagy-deficient osteoblasts from knockout mice. When inhibiting autophagy with 3 MA in wild-type osteoblasts, we found similar results as we did in autophagy-deficient osteoblasts. We also found that mechanical loading-induced ATP release is able to regulate ERK1/2, Runx2, alkaline phosphatase, and osteopontin activities. These results suggest that the ATP pathway may play an important role in the possible involvement of autophagy in osteoblast mechanobiology.

Information available at present documents the existence of three well-defined classes of guanine nucleotide binding proteins functioning as signal transducers: Gs and Gi which stimulate and inhibit adenylate cyclase, respectively, and transducin which transmits and amplifies the signal from light-activated rhodopsin to cGMP-dependent phosphodiesterase in ROS membranes. Go is a fourth member of this family. Its function is the least known among GTP binding signal transducing proteins. The family of G proteins has a number of properties in common. All are heterotrimers consisting of three subunits, alpha, beta, and gamma. Each of the subunits may be heterogeneous depending on species and tissue of origin and may be posttranslationally modified covalently. The alpha subunits vary in size from 39 to 52 kDa. The sequences for Gs alpha and transducin alpha have 42% overall homology and those of Gi alpha and Gs alpha 43%, whereas those of Gi alpha and transducin alpha have a higher degree (68%) of homology. All alpha subunits bind guanine nucleotides and are ADP-ribosylated by either pertussis toxin (Gi, transducin, Go) or cholera toxin (Gs, Gi, transducin). Thus, transducin and Gi, which have the highest degree of sequence homology, are also ADP-ribosylated by both toxins. The beta subunits have molecular weights of 36 and 35 kDa, respectively. While Gs, Gi, and Go contain a mixture of both, transducin contains only the larger (36-kDa) beta-polypeptide. The relationship of the 36- and the 35-kDa beta subunits is not defined. Although the complete sequence of the 36-kDa beta subunit of transducin has been deduced from the cDNA sequence, complete sequences of other beta subunits are not yet available so that detailed comparisons cannot be made at present. However, the proteolytic profiles of each class of the beta subunits of different G proteins are indistinguishable. The gamma subunit of bovine transducin has been completely sequenced. It has a Mr of 8400. Again complete sequences of other gamma subunits are not yet available. While the gamma subunits of Gs, Gi, and Go have identical electrophoretic mobility in SDS gels, they differ significantly in this respect from the gamma subunit of transducin. Moreover, crossover experiments point to functional differences between gamma subunits from G protein and transducin complexes. In addition, a role for beta, gamma in anchoring guanine nucleotide binding proteins to membranes has been postulated.(ABSTRACT TRUNCATED AT 400 WORDS)

The beta gamma subunits of guanine nucleotide binding proteins from bovine brain and bovine rod outer segments have different structural and immunochemical properties. In spite of these structural differences, beta gamma subunits from these sources have been found to be fully interchangeable in terms of their interaction with alpha subunits of pertussis-toxin-sensitive G proteins. In contrast, however, there are striking differences between these beta gamma subunits with regard to their ability to deactivate fluoride-stimulated Gs. These profound differences were also observed when the interaction of the purified components of the adenylate cyclase system was studied after reconstitution into phospholipid vesicles. Addition of beta gamma purified from bovine brain to vesicles containing beta-receptor and Gs results in a biphasic effect on receptor-stimulated GTPase activity, whereas addition of transducin beta gamma was virtually without any effect. Likewise, beta gamma from bovine brain, but not transducin beta gamma, affected adenylate cyclase activity of a reconstituted system consisting of three purified components (R, Gs, C). Thus, the alpha subunit of Gs, but not the alpha subunits of pertussis-toxin-sensitive G proteins discriminate between structurally different beta gamma subunits.

We have previously described a specific protease in turkey erythrocytes that converts the larger 50-kDa (P50) form of the beta 1-adrenoceptor to a smaller 40-kDa (P40) form [Jürss, R., Hekman, M., & Helmreich, E. J. M. (1985) Biochemistry 24, 3349-3354]. Further functional and structural characterization studies of the two forms are reported here. When purified P50 and P40 receptors were compared with respect to their relative capabilities to couple in lipid vesicles with pure stimulatory G-proteins (Gs-proteins) prepared from turkey erythrocytes or rabbit liver, a faster and larger activation of Gs-proteins was observed in response to l-isoproterenol and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) with P40 than with P50 receptor. The kon values for P40 were 0.47 min-1 in the case of liver Gs and 0.22 min-1 in the case of erythrocyte Gs, whereas the corresponding values for P50 were 0.34 min-1 and 0.12 min-1, respectively. The binding properties of P50 and P40 forms of the receptor were not different, and desensitization of turkey erythrocytes on exposure to l-isoproterenol did not activate the protease. We furthermore ascertained that only the larger form with a molecular mass of 50 kDa carries the N-linked carbohydrates, which are removed on proteolytic conversion to the 40-kDa form and have either a triantennary or a tetraantennary nonfucosylated complex-type structure containing terminal sialyl residues.

The properties of a reconstituted signal transmission chain using purified beta 1-adrenoceptor (R), G-protein subunits (G) and adenylate cyclase (C) in lipid vesicles are described. This assay system was used to test beta, gamma-subunits of different origin with respect to their effects on R X G and R X G X C coupling and on the functional properties of GS alpha and Gi alpha. The findings reported here point to large differences in the efficacy of beta, gamma-subunits from different sources assessed by deactivation of [ALF4]-activated rabbit liver GS and pertussis toxin-catalyzed ADP-ribosylation of bovine neutrophil G alpha. This is explained by differences in the interaction domains of the interacting subunits. Furthermore, the sensitivity of R X G and R X G X C coupling to inhibition by beta, gamma-subunits was greater than the effects of beta, gamma-subunits on hormonally activated GTPase activity of GS. One of the consequences of differential inhibition of R X G X C coupling is an amplified response of the signal transmission chain to hormonal activation. This is in agreement with observations reported by Cerione et al.