Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region

In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His1,Arg12]-, [Phe1,Arg12]-, [D-Ala4,Arg12]-, and [D-Phe4]glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [3-Me-His1,Arg12]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe1,Arg12]glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D-Ala4,Arg12]glucagon and [D-Phe4]glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala4,Arg12]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe4] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of the 10-13 region of glucagon for its receptor interactions and activation of adenylate cyclase

The role of the Tyr10-Ser11-Lys12-Tyr13 region of glucagon in the binding interaction and activation of the glucagon receptor was investigated by means of the synthetic glucagon analogues [Phe13]glucagonamide, [Phe10]glucagonamide, [Phe10]glucagon, [Phe10,13]glucagon, [Pro11]glucagon, [Pro11,Gly12]glucagonamide, [Ala11]glucagon, and [Oac11-13]glucagonamide. These analogues were synthesized by solid-phase peptide synthesis on p-methylbenzhydrylamine or Merrifield resins with protected N alpha-tert-butyloxycarbonyl amino acids. Purification by dialysis, cation-exchange chromatography, gel filtration, and preparative reverse-phase high-performance liquid chromatography (HPLC) gave products that proved homogeneous by thin-layer chromatography and HPLC and on analysis by amino acid analysis, by sequencing, and by alpha-chymotryptic peptide mapping with HPLC. Biological activities were examined by measurement of the stimulation of liver plasma membrane adenylate cyclase and by specific displacement of [125I]glucagon from glucagon receptors. The results of these studies indicate that while the biological "message" region of glucagon is located elsewhere, the 10-13 region has multiple roles in the glucagon-glucagon receptor interaction: this region provides functional groups for direct binding interaction with the receptor, and this region interacts with the receptor in such a way as to allow the "transduction message" portion of glucagon to interact and activate the receptor.     

The lipid environment of the glucagon receptor regulates adenylate cyclase activity.

1. The lipids composition of rat liver plasma membranes was substantially altered by introducing synthetic phosphatidylcholines into the membrane by the techniques of lipid substitution or lipid fusion. 40-60% of the total lipid pool in the modified membranes consisted of a synthetic phosphatidylcholine. 2. Lipid substitution, using cholate to equilibrate the lipid pools, resulted in the irreversible loss of a major part of the adenylate cyclase activity stimulated by F-, GMP-P(NH)P or glucagon. However, fusion with presonicated vesicles of the synethic phosphatidylcholines causes only small losses in adenylate cyclase activity stimulated by the same ligands. 3. The linear form of the Arrhenius plots of adenylate cyclase activity stimulated by F- or GMP-(NH)P was unaltered in all of the membrane preparations modified by substitution or fusion, with very similar activation energies to those observed with the native membrane. The activity of the enzyme therefore appears to be very insensitive to its lipid environment when stimulated by F- or gmp-p(nh)p. 4. in contrast, the break at 28.5 degrees C in the Arrhenius plot of adenylate cyclase activity stimulated by glucagon in the native membrane, was shifted upwards by dipalmitoyl phosphatidylcholine, downwards by dimyristoyl phosphatidylcholine, and was abolished by dioleoyl phosphatidylcholine. Very similar shifts in the break point were observed for stimulation by glucagon or des-His-glucagon in combination with F- or GMP-P(NH)P. The break temperatures and activation energies for adenylate cyclase activity were the same in complexes prepared with a phosphatidylcholine by fusion or substitution. 5. The breaks in the Arrhenius plots of adenylate cyclase activity are attributed to lipid phase separations which are shifted in the modified membranes according to the transition temperature of the synthetic phosphatidylcholine. Coupling the receptor to the enzyme by glucagon or des-His-glucagon renders the enzyme sensitive to the lipid environment of the receptor. Spin-label experiments support this interpretation and suggest that the lipid phase separation at 28.5 degrees C in the native membrane may only occur in one half of the bilayer.     

Gossypol inhibition of adenylate cyclase

Gossypol, a polyphenolic binaphthalene -dialdehyde reputed to exert contraceptive action in males, reversibly inhibits adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] in a concentration-dependent manner. In membranes prepared from a variety of organs, the half-maximal inhibitory concentration (IC50) ranges from 75 microM (rat Leydig tumor cells) to 250 microM (rat liver membranes). Kinetic studies using partially purified catalytic subunit isolated from bovine testis show that gossypol is competitive with ATP with an apparent Ki of 110 microM. These data suggest that gossypol inhibition of adenylate cyclase is due to direct interaction at the nucleotide-binding domain of the catalytic subunit of the enzyme.     

Melittin interactions with adenylate cyclase

Melittin, a basic polypeptide from bee venom, inhibits basal and thyrotropin-stimulated adenylate cyclase of beef thyroid membranes with a K_i ≈ 10 μM. Although this property resides in the basicC-terminal and not the N-terminal portion of the molecule, inhibition is due primarily to its detergent-like nature rather than the charge effects. There is also a small enhancing effect of both basal and thyrotropin-stimulated adenylate cyclase of 0.3–3 μM melittin.     

Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states.

Adenylate cyclase in liver membranes was solubilized with Lubrol PX and partially purified by gel filtration. The partially purified enzyme was susceptible to activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Studies on the binding of [3H]Gpp(NH)p to various fractions eluted from the gels revealed that an upper limit of 1% of the Gpp(NH)p binding sites is associated with adenylate cyclase activity stimulated by the nucleotide. The glucagon receptor, pretagged with 125I-glucagon in the membranes, solubilized with Lubrol PX, and fractionated on the same gel columns, eluted in a peak fraction that overlaps with, but is separate from, adenylate cyclase in its Gpp(NH)p-stimulated form. Addition of GTP to the solubilized glucagon-receptor complex caused complete dissociation of the complex, as has been shown with the membrane-bound form of the complex. Since the GTP-sensitive form of the glucagon receptor complex separates from the Gpp(NH)p-sensitive form of adenylate cyclase, it is concluded that the receptor and the enzyme are separate molecules, each associated with a distinct nucleotide regulatory site or component. These findings are discussed in terms of the possible structure of the hormone-sensitive state of adenylate cyclase.     

Glucagon1-6 binds to the glucagon receptor and activates hepatic adenylate cyclase.

A fragment of glucagon encompassing its first six NH2-terminal residues (His-Ser-Gln-Gly-Thr-Phe) binds to the glucagon receptor and stimulates adenylate cyclase activity in rat liver plasma membranes. Glucagon1-6 is a partial agonist since it stimulates, at saturating concentrations, to the extent of 75% of the maximal activity given by the native hormone. The binding affinity and potency of glucagon1-6 are 0.001% the native hormone. Discussed are the implications of these findings on the structure-function relationships required for the action of glucagon and for preparing clinically useful analogs of the hormone.     

Adenosine interactions with thyroid adenylate cyclase

Adenosine and certain adenosine analogues inhibit beef thyroid membrane adenylate cyclase. The inhibition has a rapid onset, is not directly on the catalytic or nucleotide regulatory sites, occurs with all activators tested (ITP, Gpp(NH)p, TSH, and F^?), and is seen also in mouse and human thyroid membranes. Addition of manganous ion, which activates adenylate cyclase, markedly enhances the inhibition by adenosine analogues. The order of potencies is: 2′,5′-dideoxyadenosine > 5′-deoxyadenosine > 2′-deoxy-3′-phosphoadenosine > 2′-deoxyadenosine > adenosine > adeninexyloside > adenine arabinoside. Purinemodified analogues are either inactive or stimulate slightly at high concentrations. This chemical specificity, the Mn²⁺ requirement, and the lack of reversal by theophylline, suggest that these membranes have little “R” site activity (stringent for the ribose moiety) and primarily contain a “P” site that has stringent purine requirement but permits changes in the ribose moiety. This site appears to be associated with the catalytic unit since it persists in solubilized adenylate cyclase.     

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.     

Alpha-adrenergic inhibition of renal cortical adenylate cyclase

Adenylate cyclase in homogenates of rat renal cortex was inhibited by alpha-adrenergic agonists. Inhibition required sodium ion and GTP. A maximum inhibition of 17.8 +/- 1.4% (S.E.M.) was produced by l-epinephrine in the presence of 0.2 M NaCl, 10 microM GTP and 10 microM propranolol. Similar inhibition was produced by l-norepinephrine and alpha-methylnorepinephrine. The EC50 values for l-epinephrine, l-norepinephrine and alpha-methylnorepinephrine were respectively 1.9 +/- 0.7 microM, 2.3 +/- 1.6 microM and 5.1 +/- 1.8 microM. Clonidine was a partial agonist causing 50% as much inhibition as epinephrine. Phenylephrine and methoxamine did not inhibit at concentrations up to 100 microM. Micromolar concentrations of phentolamine and yohimbine prevented the inhibition of adenylate cyclase by epinephrine. However, prazosin was ineffective. Thus the adenylate cyclase coupled alpha-receptors have alpha-2 specificity. Inhibition of adenylate cyclase by alpha-adrenergic agonists was not observed in homogenates of renal medulla.     

Asolectin decreases progesterone inhibition of oocyte adenylate cyclase

Asolectin, a naturally occurring mixture of soybean phospholipids, causes a small (10-25%) but reproducible stimulation on the activity of oocyte adenylate cyclase measured in the presence of guanine nucleotides. This phospholipid preparation, at a concentration of 0.5-1 mg/ml, greatly reduces the inhibition of this enzyme caused by progesterone. The reversal of the progesterone inhibition is due in part to the effect of asolectin in reducing the hysteresis of the enzymatic reaction which is enhanced by progesterone. The asolectin effect is not due to trapping of the progesterone and making it unavailable for interaction with its receptor, because similar concentrations of the phospholipid do not prevent the induction of meiotic maturation of the oocytes by progesterone. This finding suggests that the phospholipids interfere with the transduction of the hormonal signal to the adenylate cyclase catalytic subunit.     

The glucagon receptor of rat liver plasma membrane can couple to adenylate cyclase without activating it.

1. Activation of adenylate cyclase in rat liver plasma membranes by fluoride or GMP-P (NH)P yielded linear Arrheniun plots. Activation by glucagon alone, or in combination with either fluoride or GMP-P(NH)P resulted in biphasic Arrhenius plots with a well-defined break at 28.5 +/- 1 degrees C. 2. The competitive glucagon antagonist, des-His-glucagon did not activate the adenylate cyclase but produced biphasic Arrhenius plots in combination with fluoride or GMP-P(NH)P. The break temperatures and activation energies were very similar to those observed with glucagon alone, or in combination with either fluoride or GMP-P(NH)P. 3. It is concluded that although des-His-glucagon is a potent antagonist of glucagon, it nevertheless causes a structural coupling between the receptor and the catalytic unit.