Ernst J.M. Helmreich

Hormone-receptor-adenylate cyclase interactions

Since p~iI~Iicat~on of the first review tetter [I J on hormone receptor interactions in January 1976 progress in the field has been rapid. Recent advances now make it possible to discuss hormone-receptor-ade~ylate-cyciase interactions and their control in more definitive terms. This has encouraged us to present some further thoughts on the same topic. A comprehensive coverage of the literature was not intended and certainly the selection of topics is strongly biased by the authors interests. A large number of hormones and neurotransmitters activate adenylate cyciase (EC 4.6.1.1) in the course of eliciting their physiological response. In each case the hormone or the neurotransrnitter interacts with a specific receptor at the outer surface of the cell which, in turn, interacts with the catalytic moiety of adertylate cyclase that faces the inside of the cell and causes its activation:

Structural and Functional Relationships of Guanosine Triphosphate Binding Proteins

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)

Signal Transfer from Hormone Receptor to Adenylate Cyclase

Publisher Summary This chapter discusses signal transfer from hormone receptor to adenylate cyclase. One of the fundamental properties of living systems is their capacity to receive and to respond to signals. Isolation and purification of a specific hormone-binding protein from membranes to a state of apparent homogeneity has been achieved only in the case of the insulin receptor of fat and liver cell membranes and of human lymphocytes. Nonionic detergents were used for solubilization and affinity chromatography with tightly binding ligands for purification. Considerable progress has been made in the isolation and characterization of specific membrane-binding proteins for insulin and acetylcholine. However, no assay is yet available to test the biological function of the isolated hormone receptor, although a plausible relationship has been established between insulin binding and biological response in intact cells. One of the serious shortcomings in catecholamine-binding experiments is the relatively low affinity of this group of hormones to membrane sites.

A carbene-generating photoaffinity probe for beta-adrenergic receptors

A new radioiodinated (2.2 Ci/mumol) iodocyanopindolol derivative carrying a 4-(3-trifluoromethyldiazirino)benzoyl residue has been synthesized. The long-wavelength absorption of the diazirine permits formation of the carbene by photolysis under very mild conditions. [125I]ICYP-diazirine binds with high affinity (Kd = 60 pM) to beta-receptors from turkey erythrocyte membranes. Upon irradiation, [125I]ICYP-diazirine is covalently incorporated in a Mr 40 000 protein. Stereoselective inhibition of photolabeling by the (-)enantiomers of alprenolol and isoproterenol indicated that the Mr 40 000 protein contains a beta-adrenergic binding site. The yield of specific labeling was up to 8.2% of total beta-receptor binding sites. The Mr 40 000 protein photolabeled in the membrane could be solubilized at comparable yield with either digitonin or Triton X-100. Irradiation of digitonin-solubilized turkey erythrocyte membranes with [125I]ICYP-diazirine resulted in specific labeling of two proteins with Mr 40 000 and 50 000. In guinea-pig lung membranes, at least five proteins were photolabeled, of which one (with approximately Mr 67 000) was labeled specifically.

Interactions of pure βγ-subunits of G-proteins with purified β1-adrenoceptor

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.

A proton donor-acceptor function of the 5'-phosphate group of pyridoxal-P in potato phosphorylase inferred from 31P NMR spectra.

Pyridoxal-

Fluorescent glucagon derivatives. I. Synthesis and characterisation of fluorescent glucagon derivatives

-phosphate is essential for the activity of prokaryotic and eukaryotic cy-glucan phosphorylases (EC 2.4.1 .l) [1,2]. For expression of activity, mammalian phosphorylases [2] must be activated either allosterically by 5’-AMP or covalently through phosphorylation of a seryl residue, whereas phosphorylases from potato tubers [3] (and Escherichia coli [4,5] or Klebsiella pneumoniae [6]) are neither subject to allosteric nor covalent control. Despite these differences there is a high degree of homology in the sequence around the pyridoxal-5’-phosphate binding site [ 1,5]. This makes potato phosphorylase ideally suited for 31P NMR studies on the role of the cofactor in catalysis.

Synthesis and characterization of CGP-12177-NBD: a fluorescent β-adrenergic receptor probe

The synthesis of monofluorescein, monorhodamine, and mono-4-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives of glucagon is reported. The fluorescent groups were introduced by converting tryptophan-25 to 2-thioltryptophan using thiol-specific fluorescent reagents. All derivatives retained the ability to activate adenylate cyclase when compared to glucagon and thus were considered full agonists. IC50 values of 6.8.10(-9), 1.7.10(-8), 1.8.10(-8) and 5.4.10(-9) M were measured in rat liver membranes for NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. From the IC50 values Kd values of 2.16.10(-9), 4.10(-9), 2.10(-9) and 1.72.10(-9) M were calculated for the binding of NBD-, fluorescein-, rhodamine-Trp25-glucagon and native glucagon, respectively. The highest quantum yield (0.18) of the monomer derivatives was obtained with fluorescein-Trp25-glucagon in phosphate-buffered saline (pH 7.4). Difluorescein-glucagon was also prepared by reacting the amino groups of histidine-1 and lysine-12 with fluorescein isothiocyanate and dimer derivatives were prepared using fluorescein-labelled 2-thiolTrp25-glucagon. Difluorescein-glucagon bound only weakly to glucagon receptors and displayed antagonist properties. The dimer derivative formed from two difluorescein-2-thiolTrp25-glucagon molecules had similar poor binding qualities, whereas the dimer formed from difluorescein-2-thiolTrp25-glucagon and 2-thiolTrp25-glucagon exhibited, at low concentrations, properties similar to monofluorescein-glucagon. Both dimer derivatives were only sparingly soluble in aqueous medium. Specific binding of fluorescein-Trp25-glucagon and difluorescein-glucagon to rat hepatocytes was followed using flow cytometry.

Is the active form of pyridoxal-P in α-glucan phosphorylases a 5′-phosphate dianion?

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.

Fluorescent glucagon derivatives. II. The use of fluorescent glucagon derivatives for the study of receptor disposition in membranes

The essential role of pyridoxal-5’-phosphate for the activity of all cY-glucan phosphorylases received strong support from recent studies with bacterial phosphorylases [ 11. The advantage of utilizing these enzymes for the study of the role of pyridoxal-P is obvious since they neither depend on allosteric effectors or covalent modification for the expression of activity [l-6]. The 5’-phosphate group of pyridoxalP is the sole phosphate moiety in catalytically active bacterial phosphorylases. Accordingly, ‘rP NMR spectra of Escherichia coli maltodextrin phosphorylase exhibit an exceptionally simple pattern. When compared with the 31P NMR spectra of rabbit skeletal muscle phosphorylase a and b [7] the resonances common to phosphorylase allow us to define the ionization state of the cofactor in the catalytically active enzyme.