Stabilization of soluble active rat liver glucagon receptor.

Active glucagon receptor was solubilized with 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (Chaps) from rat liver plasma membranes but rapidly (less than 8 h) lost activity. Either inclusion of 1X Hanks' balanced salt solution in the 3 mM Chaps solubilization buffer or its addition after solubilization increased the percentage of total binding attributable to specific glucagon binding from approximately 10 to greater than 80%; of great importance, it increased the stability from near zero binding at 8 h to 50% binding at 48 h (4 degrees C). Of the Hanks' solution components, either NaCl (137 mM) or CaCl2 (1.26 mM) was effective in increasing specific binding to approximately 70 and 60% respectively: Mg salts were ineffective. Soluble receptor binding activity was assayed by dextran-coated charcoal adsorption of free hormone. The assay is rapid, simple, and reproducible. It is suitable for monitoring receptor activity during purification and molecular characterization. Competition binding studies gave an IC50 value of 10-20 nM (slope factor approximately 1), with or without GTP. Dissociation assays revealed GTP sensitivity when receptors were solubilized either as glucagon-receptor complexes or free receptor. Active glucagon-receptor complexes could be eluted from wheat germ lectin-agarose: neither concanavalin A-agarose nor soybean agglutinin-agarose bind receptor. A glucagon degrading activity which co-solubilized with the receptor but did not require detergent for extraction was distinguishable from the soluble receptor not only by solubility but also by its heat stability (30 degrees C), its inhibition by bacitracin, its affinity for glucagon, its retention of activity for at least 1 week at 4 degrees C, and its size.

Quantitative analysis of internalization of glucagon by isolated hepatocytes.

Biochemical methods have been used to quantitate total, acid-stable and acid-labile association of (mono[125I]iodoTyr10) glucagon with rat hepatocytes in suspension to evaluate internalization of glucagon and its receptors. Internalization is inhibited by low temperature, phenylarsine oxide, and by blocking receptor binding, consistent with receptor-mediated endocytosis. Approximately 30% of the total cell-associated hormone is internalized at 30 min of incubation. The rate declines until 90 min when the internalization of glucagon ceases, although the cells remain competent to internalize asialofetuin. From 90 min to 4 h, 27% of the maximum label internalized at 30 min remains within cells. The number of cell surface receptors decreases but the affinity of those remaining is unchanged. However, 1.7-2.7 surface receptors are lost to binding for each molecule of radiolabeled glucagon internalized. Uptake occurs according to a rate constant of 0.183 min-1 (t1/2 = 3.8 min). We conclude that (i) hepatocytes internalize a finite quantity of glucagon, implying the existence of undefined regulatory mechanisms; (ii) hormone is retained for greater than 2 h within cells and may play a physiological role within cells; and (iii) both occupied and unoccupied receptors become inaccessible to extracellular hormone as internalization proceeds; rapid recycling of receptors does not occur.

Guanine nucleotide regulation of the interconversion of the two-state hepatic glucagon receptor system of rat.

To investigate whether guanine nucleotides regulate interconversion of the two-state hepatic glucagon receptor we have utilized kinetic assays of glucagon binding to partially purified rat liver plasma membranes. Dissociation of glucagon at 30 degrees C exhibited biexponential character in either the absence or presence of GTP, indicating that the system previously seen in intact hepatocytes is independent of intracellular modulators. In each case the receptors underwent a time-dependent conversion from a low affinity to a high affinity state. However, GTP decreased the fraction of receptors in the high affinity state. The rank order for stabilizing the low affinity state was Gpp(NH)p greater than GTP greater than GDP much greater than GMP = no nucleotides. Data from competition binding assays with increasing concentrations of GTP allow calculation of equilibrium constants which are 3.32 nM for glucagon and receptor in the absence of GTP, 18.6 nM for glucagon and receptor in the presence of GTP, 1.55 microM for the association of receptor and GTP presumably linked to an N protein, and 8.86 microM for the association of the glucagon-receptor complex and GTP again presumably linked to an N protein, Glucagon binding to receptor is noncooperative in both the absence and presence of GTP, distinguishing this system from the beta-adrenergic system. With GTP, binding to the low affinity state is favored because of the relative affinities reported. Therefore, GTP regulates the activation by slowing the conversion of the receptor from a low affinity to high affinity form.

Semisynthetic D-His1,N epsilon-acetimidoglucagon: structure-function relationships.

The histidine residue at the amino terminus of lysine-12 protected glucagon was replaced by its D-isomer by an established semisynthetic strategy to extend a stepwise series of replacements at this position. The product was examined for its secondary structure and its function. Circular dichroism spectra obtained at concentrations from 0.25 to 1.09 mg/ml at pH 10.2 in 0.2 M phosphate buffer were similar to those obtained with native hormone. Competitive binding assays and adenylate cyclase activation assays with partially purified rat liver plasma membranes show this D-His1 analog of glucagon to be a full agonist, causing the same maximum activation of adenylate cyclase as native hormone; but both binding and activation assays show the binding affinity to be diminished about 10-fold. The data suggest that the adjustment of the bonding of the imidazole group to the receptor to bring about transduction results in constraints on the conformation along the peptide sequence which interfere with the peptide adopting the same binding conformation achieved by the native hormone.

Biological activities of catfish glucagon and glucagon-like peptide.

The ability of catfish glucagon and glucagon-like peptide to bind and activate mammalian glucagon receptors was investigated. Neither catfish peptide binds to glucagon receptors of rat liver, hypothalamus or pituitary. Neither stimulates adenylate cyclase activity in liver membranes. Catfish glucagon fails to activate adenylate cyclase in hypothalamic or pituitary membranes in contrast to mammalian glucagon. However, catfish glucagon-like peptide does stimulate hypothalamic and pituitary adenylate cyclase (EC50 approximately 1 pM) possibly through mammalian glucagon-like peptide receptors.

Partial agonism in the glucagon receptor system is a consequence of the two-state rat hepatic receptor.

We have previously demonstrated that the glucagon receptor binds hormone to form a low affinity complex which, by a time- and temperature-dependent mechanism, is converted to a high affinity complex (Horwitz, E.M., Jenkins, W.T., Hoosein, N.M., and Gurd, R.S. (1985) J. Biol. Chem. 260, 9307-9315). In this report we have investigated the effects of agonist concentration, potency, and intrinsic activity on the characteristics of the two, interconvertible states of the glucagon receptor. As the glucagon concentration is increased from 0.02 to 0.50 nM, the initial velocity of binding increases. The conversion of a low affinity to a high affinity complex is the rate-limiting step in the overall binding reaction and approaches its maximal velocity as the hormone concentration exceeds 0.20 nM. At equilibrium, 87-90% of the hormone-receptor complexes are in the high affinity state at all hormone concentrations examined. [S-methyl-Met27]glucagon, a full agonist with reduced potency, binds to the two-state system in a manner analogous to that of native glucagon. The binding of N alpha-biotinyl-N epsilon-acetimidoglucagon, a partial agonist with reduced potency, effects a two-state system where the high affinity state accounts for only 35% of the total hormone-receptor complexes at equilibrium. We conclude that the formation of the high affinity complex is the rate-limiting step involved in glucagon binding; reduction in binding potency with full agonism is due to a reduction in the affinity of the ligand for the unoccupied receptor and not to an alteration of the interconversion of the two states, and decreased intrinsic activity is due to a quantitative decrease in conversion of the low to high affinity state.

Electrostatic interactions in sperm whale myoglobin. Site specificity, roles in structural elements, and external electrostatic potential distributions.

The electrostatic free energy contribution to the stability of sperm whale ferrimyoglobin was evaluated according to the static accessibility modified Tanford-Kirkwood model. The electrostatic free energy contribution of each distinct structural element was divided into one term arising from interactions between it and other elements (interelemental) and another from interactions within the particular element itself (intraelemental). At pH 7 the majority of the terms were found to be stabilizing. The interelemental terms are the dominant ones for most structural elements. The small interelemental terms of the C and D helices are compensated by large intraelemental interactions which stabilize these short helices. Perturbations in pH can be accommodated by the structural elements through a redistribution of stabilizing and destabilizing interactions. The electrostatic potentials calculated at the surface of the protein indicate that the internal compensation of local potentials achieved during folding results in a generally neutral protein-solvent interface save for two distinct areas of nonzero potential. The accessibility of each charged atom to solvent was analyzed in terms of the surface area lost to charged, polar and nonpolar atoms separately. The net solvent accessibility lost parallels closely that lost to nonpolar atoms alone, indicating a specific role for nonpolar atoms in defining dielectric shielding of charged atoms, aside from their participation in the well-known hydrophobic interactions.

Kinetic identification of a two-state glucagon receptor system in isolated hepatocytes. Interconversion of homogeneous receptors.

A detailed kinetic study was performed to investigate the interaction of glucagon with receptors on freshly isolated hepatocytes. Competition binding assay results fit a mathematical expression for a single site noncooperative model of binding. Glucagon was shown to bind with first-order kinetics at six-hormone concentrations (0.02-0.50 nM) at 0 and 37 degrees C. The observed pseudo-first-order rate constants are directly proportional to the hormone concentration at 0 degree C, but display a downward deviation from linearity at 37 degrees C. Dissociation of glucagon exhibited biexponential character at 37 degrees C which was not seen at 0 degree C. The biphasic dissociation at 37 degrees C was resolved into rapid (t1/2 = 1.9 min) and slow (t1/2 = 27.7 min) components. The distribution of the total bound hormone between the rapidly and slowly dissociating complexes was not dependent upon the extent of receptor occupancy. The absolute quantity of rapidly dissociating hormone-receptor complexes was constant at all times examined; however, the fraction of slowly dissociating hormone-receptor complexes was found to increase with increasing incubation time. The results indicate that a homogeneous population of hepatic receptors undergoes a time-dependent, temperature-dependent conversion from one state to another in a two-stage sequential manner.

Structure-function relationships of S-carboxymethyl methionine27 glucagon.

Carboxymethylation of glucagon and subsequent purification of the hormone has provided a derivative modified by the addition of bulk to the methionine at position 27 without a net charge alteration in the side chain. Unreacted glucagon was removed after methylation of the methionine which provides a positively charged chromatographic handle. The derivative has a half-maximum concentration for binding of 5.3 nM and is a full agonist. These findings along with those provided by methylation of the methionine indicate that a positive charge rather than bulk on the methionine side chain disrupts the binding of hormone to its receptor. The S-carboxymethyl derivative lacks the concentration-dependent aggregation characteristic of glucagon at pH 10.2 as does the S-methyl derivative but increases its helical content in 30% 2-chloroethanol to the same extent as native and S-methyl hormone. Full activity of the S-carboxymethyl methionine27 glucagon does not favor the existence of the globular structure proposed by Korn and Ottensmeyer [(1983) J. Theor. Biol. 105, 403] as the binding species whereas multiple considerations do favor a flexible hormone with nucleation followed by conformational changes for complete binding and activation. Isotopic enrichment using labeled iodoacetate is feasible and can provide more definitive structural information.

Human glucagon-like peptides 1 and 2 activate rat brain adenylate cyclase.

Two human glucagon-like peptides, GLP-1 and GLP-2, which are coencoded with pancreatic glucagon in the preproglucagon gene, do not significantly inhibit [125I]monoiodoglucagon binding to rat liver and brain membranes and do not activate adenylate cyclase in liver plasma membranes. Nevertheless, GLP-1 and GLP-2 were each found to be potent stimulators of both rat hypothalamic and pituitary adenylate cyclase. Only 30-50 pM concentrations of each peptide elicited half-maximal adenylate cyclase stimulation. Our data suggest that GLP-1 and GLP-2 may be neurotransmitters and/or neuroendocrine effectors, which would account for their high degree of sequence conservation through vertebrate evolution.

N alpha-Malto-glucagon and N alpha-malto, S-methyl methionine27-glucagon: preparation and characterization of two partial agonists.

N alpha-Maltoglucagon was prepared by demethylation of N alpha-malto, S-methyl methionine27 glucagon, and the two derivatives were purified to greater than 99% and 99.7%, respectively. S-Methylation of glucagon lowers the reactivity of Lys-12 and provides an alternative strategy to epsilon-amino protection for directing glycosylation of glucagon to the alpha-amino group. Both derivatives are partial agonists, with their adenylate cyclase activation and binding reduced in parallel. N alpha-Maltoglucagon produces 70% and N alpha-malto, S-methyl methionine27 glucagon 40% of the maximum activity of native hormone. N alpha-Maltoglucagon binds equivalently to N alpha-biotinyl, N epsilon-acetimidoglucagon whose maximum activity is near 35%, but a pK shift of the imidazole moiety cannot account for the difference in their abilities to produce transduction. Both glycosylated derivatives bind noncooperatively and both inhibit adenylate cyclase at high concentrations. The presence of a maltose residue on the amino terminal of glucagon may be required but, alone provides insufficient structural complementarity for concanavalin A binding to occur. The glycosylated derivatives are resistant to aminopeptidase degradation, are more soluble, and the maltose residue is unlikely to cause toxicity with in vivo use. Such attributes may be advantageous in the development of other analogs.

Identification of glucagon receptors in rat brain.

The binding of radiolabeled glucagon to rat brain membranes was investigated. Regional distribution studies indicate higher specific binding of 125I-labeled monoiodoglucagon to olfactory tubercule, hippocampus, anterior pituitary, and amygdala membranes, with somewhat lower binding to membranes from septum, medulla, thalamus, olfactory bulb, and hypothalamus. 125I-labeled glucagon bound to rat brain synaptic plasma membrane fractions with high affinity (KD = 2.24 nM). Specific binding was greater to synaptosomal membrane fractions relative to myelin, mitochondrial nuclear, or microsomal fractions. Inclusion of 0.1 mM GTP in the binding assay reduced the glucagon binding affinity (KD = 44.5 nM). Several neuropeptides and other neuroactive substances tested did not affect binding of labeled glucagon to brain membranes. Three different glucagon analogs inhibited labeled glucagon binding. Synthetic human pancreatic growth hormone-releasing factor, hpGRF-44, also inhibited binding, although the concentration required for half-maximal displacement was 100-fold higher than for native glucagon. Addition of glucagon to brain membranes resulted in approximately equal to 3-fold maximal activation of adenylate cyclase over basal levels. Glucagon at a concentration of 4.74 nM was required for half-maximal activation of pituitary membrane adenylate cyclase. These findings provide evidence for rat brain binding sites that respond to the pancreatic form of glucagon and can transduce this binding into the activation of adenylate cyclase.

Semisynthetic derivatives of glucagon. The contribution of histidine-1 to hormone conformation and activity.

Semisynthetic N epsilon- acetimidoglucagon was prepared from the [des- His1 ]analogue by coupling the N-hydroxysuccinimide ester of N alpha- tBoc - Nimidazole -DNP-L-histidine to the peptide in dimethylformamide in the presence of 1-hydroxybenzotriazole. The deprotected, purified product was chemically identical to N epsilon- acetimidoglucagon and equipotent to N epsilon- acetimidoglucagon and native glucagon in its ability to activate adenylate cyclase and displace [125I] iodoglucagon from rat liver plasma membranes. Semisynthetic [ Phe1 ]-, [ Ala1 ]-, and [des- His1 ] glucagons prepared similarly achieved 85, 55, and 35% of the maximal activity and 22, 2, and 6% of the binding potency of N epsilon- acetimidoglucagon . The biological assays indicate that the amino group is involved to a greater extent in transduction than in binding, but the aromatic nature and hydrogen bonding capability of the imidazole ring of histidine-1 are important for both binding and transduction. In circular dichroism studies, all derivatives exhibited increased helicity in 2-chloroethanol. The [ Phe1 ] analogue although less soluble behaved similarly to native glucagon, while the [ Ala1 ] and [des- His1 ] derivatives exhibited an increased helical content in 0.01 N HCl as a result of an increased propensity of these derivatives to self-associate in the absence of 2-chloroethanol. The unexpected conformational changes throughout the molecule may have relevance for the functional activity.

Noncooperative receptor interactions of glucagon and eleven analogues: inhibition of adenylate cyclase.

Glucagon and 11 glucagon derivatives were characterized and compared with respect to the cooperativity of their receptor interactions and their ability to elicit a biphasic (activation-inhibition) response from the adenylate cyclase system of rat liver plasma membranes. Slope factors were evaluated from two sets of experimental data, binding to hepatocyte receptors and activation of adenylate cyclase. The results are consistent with noncooperative binding to a single affinity state of the glucagon receptor for all derivatives, irrespective of the modification and the agonist properties of the derivatives. High-dose inhibition of adenylate cyclase activity was observed for native glucagon and all of the derivatives which were examined at high concentrations (greater than 10(-5) M). Partial agonism of some low-affinity glucagon derivatives is not caused by high-dose inhibition. Several mechanisms which might give rise to high-dose inhibition such as receptor cross-linking or multivalent receptor binding are discussed in relationship to the glucagon-receptor interaction. These phenomena indicate that significant differences exist between the glucagon system and the beta-adrenergic system.

Semisynthetic derivatives of glucagon: (des-His1)N epsilon-acetimidoglucagon and N alpha-Biotinyl-N epsilon-acetimidoglucagon.

N epsilon-Acetimidoglucagon to be used for semisynthesis was prepared by reacting glucagon with methyl acetimidate hydrochloride at pH 10.2, favoring acetimidation of the sole epsilon-amino group. N epsilon-Acetimidoglucagon was isolated from the crude acetimidoglucagon mixture by anion-exchange chromatography at pH 9.4, producing a derivative which was identical with native glucagon on isoelectric focusing and which by amino acid analysis had greater than 98% of the lysine blocked. The yield was greater than that obtained when tetrahydrophthalic anhydride was used as a chromatographic handle to remove peptides with unreacted amino groups. N epsilon-Acetimidoglucagon closely resembled native glucagon in its biological activity and binding affinity, eliminating the need for deprotection. Semisynthetic N alpha-biotinyl-N epsilon-acetimidoglucagon, prepared by reacting (N-hydroxysuccinimido)biotin with N epsilon-acetimidoglucagon and purified by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 5.2) and exhibited 1.2% of the binding affinity, 2.4% of the biological potency, and 30% of the maximum activity of the native hormone. Preliminary fluorescence microscopy demonstrated binding of N alpha-biotinyl-N epsilon-acetimidoglucagon to glucagon specific receptors following exposure to fluorescein-labeled avidin. Capping of labeled receptors could be visualized with time. (Des-His1)N epsilon-acetimidoglucagon, prepared via a manual Edman degradation of N epsilon-acetimidoglucagon and isolated by cation-exchange chromatography, was homogeneous upon isoelectric focusing (pI = 5.2). The second residue, serine, has also been removed. Semisynthetic coupling of alternative residues to such derivatives will provide insight into the role of the amino-terminal residues in mediating the biological actions of the hormone.

Glucagon carboxyl-terminal derivatives: preparation, purification, and characterization.

Chemical and enzymatic methods have been used to prepare the following series of seven glucagon derivatives modified in the carboxyl-terminal region important for hormone-receptor binding: [des-Asn28,Thr29](homoserine lactone27)glucagon, [des-Asn28,Thr29](homoserine27)glucagon, (S-methyl-Met27)glucagon, [des-Thr29](S-methyl-Met27)-glucagon, [des-Thr29]glucagon,[des-Asn28,Thr29](S-methyl-Met27)glucagon, and [des-Asn28,Thr29]glucagon. The derivatives were isolated in high yield, extensively purified, and chemically characterized. All were found to be full agonists of native glucagon. Binding affinity was evaluated by displacement of mono[125I]iodoglucagon prepared by new methods. Binding and biological activities closely correlated, indicating that most modifications affected the relative binding affinity and relative biological potency of glucagon to a comparable extent. Circular dichroism measured in dilute acid solution resembled that of native glucagon except for [des-Asn28,Thr29]glucagon which displayed increased alpha helicity (25%). All derivatives formed helical structures in 2-chloro-ethanol, although the amount of helicity induced was not closely correlated with biological activity. Binding and biological activities were not affected by removal of Thr-29, though both were reduced 20-fold when Asn-28 was also removed, irrespective of whether homoserine or native methionine remained at the carboxyl terminus. Lactone formation was associated with a further 5-fold reduction in binding affinity but not in activity. Methylation of Met-27 had essentially the same effect as removing the two carboxyl-terminal residues, although the combined effect of both modifications was greater than 100-fold reduction in binding and activity. These findings provide additional insight concerning glucagon structure-function relationships.

Electrostatic stabilization in sperm whale and harbor seal myoglobins. Identification of groups primarily responsible for changes in anchoring of the A helix.

The compact, largely helical structure of sperm whale and harbor seal myoglobins undergoes an abrupt one-step transition between pH 4.5 and 3.5 as monitored by changes in either the heme Soret band absorbance or circular dichroism probes of secondary structure, for which a modified Tanford-Kirkwood theory provides identification of certain dominant electrostatic interactions responsible for the loss of stability. A similar treatment permits identification of the electrostatic interactions primarily responsible for a process in which the anchoring of the A helix to other parts of the molecule is weakened. This process is detected with both myoglobins, in a pH range approximately 1 unit higher than the onset of the overall unfolding process, through changes in the circular dichroic spectra near 295 nm which correspond to the L1 O-O band of the only two tryptophan residues in these proteins, residues 7 and 14. In each case protonation of certain sites in neighboring parts of the molecule can be identified as producing destabilizing interactions with components of the A helix, particularly with lysine 6.

The quantitation of carbamino adduct formation of angiotensin II and bradykinin.

The two equilibrium constants that define the extent of carbamino adduct formation with amines for all values of pH and PCO2 are determined for the alpha-amino groups of the peptide hormones angiotensin II(AII) and bradykinin (BK) by nuclear magnetic resonance techniques. From these constants the variation of carbamino adduct formation has been calculated over the pH range 6.60--8.00 with variable PCO2, and the results are superimposed upon standard pH-bicarbonate diagrams. PCO2, and the results are superimposed upon standard pH-bicarbonate diagrams. The mole fraction, Z, of carbamino adduct form of AII or BK shows a maximum variation in going from metabolic alkalosis, Z congruent to 0.30, to metabolic acidosis, Z congruent to 0.02, with Z near 0.2 for normal acid-base conditions. Adduct formation to hormone may alter the biological effect of the hormone (a) by limiting proteolysis, particularly at the amino-terminal, (b) by altering hormone binding affinity to specific receptors, or (c) by converting the hormone to an antagonist which binds to receptor but does not activate subsequent metabolic events. The requirements for any of these mechanisms to operate are examined in terms of simple equilibrium considerations, and experimental evidence of inhibition of an aminopeptidase model system is presented. These results are consistent with the hypothesis that regulation of some physiological processes through formation of carbamino adduct of peptide hormones is possible.