Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin-releasing hormones in the pituitary of goldfish (Carassius auratus)

The relationship between gonadotropin-releasing hormone (GnRH) receptor binding and biological activity in the goldfish pituitary for mammalian and salmon GnRH (sGnRH) analogs with structural modification at the C terminus involving replacement of glycine amide with an alkyl amine and replacement of the Gly6 residue with D amino acids was examined. The GnRH receptor binding data were analyzed with a computerized curve-fitting program (LIGAND) for a single as well as two classes of binding sites; analysis based on one site fit estimated binding affinity and capacity for one class of binding site, and analysis based on two-site fit estimated binding affinity and capacity for two classes of binding sites (high-affinity/low-capacity and low-affinity/high-capacity binding sites). The estimated receptor affinity values were then used to determine the correlation between binding affinity and gonadotropin (GTH)-release potency in vitro. The highest correlation between biological activity and receptor binding affinity was obtained for the high-affinity/low-capacity binding sites and GnRH analogs containing Trp7 and Leu8 residues (i.e., the salmon GnRH structural format) (R = 0.940 +/- 0.150). For the same group of GnRH analogs, there was no significant correlation between the relative GTH-release potency and binding affinity of the low-affinity/high-capacity sites (R = 0.159 +/- 0.434), or that obtained from a one-site fit (R = 0.198 +/- 0.431). Similarly, for mammalian GnRH analogs, significant correlation between binding affinity and biological activity (R = 0.406 +/- 0.049) was only obtained for the high-affinity sites, although the degree of correlation was significantly lower than that obtained for salmon GnRH analogs. The present findings provide strong support for the hypothesis that high-affinity GnRH receptors are involved in the control of GTH release in the goldfish pituitary. In addition, the results demonstrate clearly that the presence of Trp7, Leu8 residues in salmon GnRH molecule, a native peptide in goldfish, is important for recognition of the ligand by the GnRH receptors in the goldfish pituitary, and that structural modifications at positions 6 and 10 in this peptide can increase receptor binding affinity and biological activity at the pituitary level. The most active sGnRH analog identified to date is [D-Arg6, Pro9-NEt]-sGnRH.     

In vitro cytokinin binding to a particulate fraction of tobacco cells

At least two types of cytokinin-binding sites are present in a particulate fraction of tobacco (Nicotiana tabacum L.) cells that sediments at 80,000 x g. The major binding component has a low affinity towards cytokinins, is resistant to heating at 100°C, and is not specific for biologically active cytokinin analogues. The second site occurs in much lower frequency, is heat labile, shows high affinity towards cytokinins, and is specific for biologically active analogs of the hormone. The testing for binding specificity was mainly performed with a series of halogenated benzyladenine derivatives having a wide range of biological activities. The low-affinity binding site shows some of the same features as talcum powder, a non-biological material which binds cytokinins in a non-specific fashion. The properties of the high-affinity binding site are consistent with the expected characteristics of a cytokinin receptor. However, the role of the observed high-affinity binding site with regard to the biological action of cytokinins is not yet known.Abbreviations BA N ⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - K_d equilibrium dissociation constant - R_t total concentration of binding sites In partial fulfillment of the requirements for the Ph.D. degree in the Department of Botany and Plant Pathology, Michigan State University     

Gonadotropin-releasing hormone receptor binding in bovine anterior pituitary

Synthetic gonadotropin-releasing hormone (GnRH) was monoiodinated at a high specific radioactivity with 125I. The iodinated hormone retained full biological activity as assessed by the release of luteinizing hormone in vitro from bovine anterior pituitary tissue slices. Specific binding of 125I-labeled gonadotropin-releasing hormone of high affinity and low capacity was obtained using dispersed bovine anterior pituitary cells. The binding had sigmoid characteristics, compatible with the presence of more than one binding site. The subcellular fraction responsible for binding was identified with the plasma membranes. However, significant binding also occurred in the secretory granules fraction. The plasma membranes were solubilized with sodium dodecyl sulfate. Using gonadotropin-releasing hormone covalently coupled to a solid phase, a protein was purified by an affinity technique from the solubilized plasma membrane preparation which possessed similar binding propperties as plasma membranes, both intact and solubilized. The protein migrated as a single component on polyacrylamide gel in sodium dodecyl sulfate and the estimated molecular weight was 60 000. The character of the gonadotropin-releasing hormone concentration dependence binding as well as association kinetics were multiphasic and suggested the presence of more than one binding site. When analyzed by the Hill plot, the Hill coefficient of all binding curves was always greater than one which is compatible with positive cooperativity. This was further supported by the dissociation studies where the dissociation rate was inversely proportionate to both the gonadotropin-releasing hormone concentration and the time interval during which the gonadotropin-releasing hormone-gonadotropin-releasing hormone receptor protein complex was formed. Using difference chromatography, aggregation of the purified gonadotropin-releasing hormone receptor protein was demonstrated to occur upon its exposure to gonadotropin-releasing hormone. The formed macromolecular complexes bound preferentially 125I-labeled gonadotropin-releasing hormone. It is concluded that a single receptor protein is responsible for gonadotropin-releasing hormone binding in the bovine anterior pituitary. It is a part of the plasma membranes. Its interaction with gonadotropin-releasing hormone provokes transitions of the protein into different allosteric forms and this may be related to the biological effect of gonadotropin-releasing hormone on gonadotropin secretion.     

Pituitary somatostatin receptors. Characterization by binding with a nondegradable peptide analogue

Somatostatin receptors in the rat pituitary gland were characterized by binding analysis with a radioiodinated high affinity somatostatin analogue, 125I-Tyr1[D-Trp8]somatostatin. Receptor binding of this derivative reached equilibrium at 30 min and was maintained at a plateau for at least 60 min. Two L-Trp8- labeled somatostatin analogues. 125I-Tyr1- and [125I-Tyr11]somatostatin, displayed less stable and lower specific uptake and higher nonspecific binding. In contrast to the rapid degradation of the L-Trp8 ligands during binding assay, 125I-Tyr1]D-Trp8]somatostatin retained more than 80% of its binding activity after 90 min of incubation with pituitary particles. Pituitary particles bound 125I-Tyr1]D-Tyr8]somatostatin with high affinity (Ka = 8.6 +/- 1.2 X 10(9) M-1) and capacity of 54.4 +/- 2.6 fmol/mg. These binding sites showed specificity for the native peptide and its active analogues, and other peptide hormones, including angiotensin II, thyrotropin-releasing hormone, vasopressin, oxytocin, substance P, and gonadotropin-releasing hormone, did not inhibit tracer binding. A good correlation was observed between the binding affinities of several somatostatin analogues and their potencies as inhibitors of growth hormone release in rat pituitary cells. These findings emphasize the physiological importance of the pituitary somatostatin receptor in mediating the inhibitory action of the peptide on growth hormone release. The use of Tyr1[d-Trp8]somatostatin as a labeled ligand permits accurate determinations of the binding affinity and concentration of receptors for somatostatin in the normal pituitary gland and provides a basis for further studies of somatostatin receptor regulation and receptor-mediated cellular effects of the tetradecapeptide.     

Biological activities of thionated thyrotropin-releasing hormone analogs.

Analogs of thyrotropin-releasing hormone (Glp-His-Pro-NH2, TRH) have been prepared which contain thioamide moieties in the pyroglutamic acid ring, the carboxyamide proline terminus, and in both positions (dithio). These compounds have been tested for TSH-releasing activities (in vitro and in vivo), and for binding to TRH receptors in rat pituitary and cortex. The monothionated analogs showed no significant differences in TSH-releasing potency from TRH either in vitro or in vivo. However, with two thioamide replacements the potency decreases about 50%. Significantly, in terms of receptor selectivity, thionation has resulted in differentiation between brain receptors (pituitary and cortex). The Pro psi[CSNH2] and dithio analogs were more selective (higher affinity to pituitary receptors) than the parent hormone, while the analog containing a thioamide replacement in the pyroglutamyl ring had lower affinity and was not selective. These results suggest that the subtle exchange of sulphur for oxygen can have an important impact on both receptor selectivity and affinity within a biologically active peptide.     

Solubilized active pituitary and ovarian gonadotropin-releasing hormone receptors retain binding properties for adenosine 3′, 5′-cyclic monophosphate derivatives

The non-denaturing zwitterionic detergent, (3 (3-cholamidopropyl)-dimethyl-ammonio)-1-propane sulfonate (CHAPS), has been used to solubilize membrane gonadotropin-releasing hormone (GnRH) receptors from rat ovaries. The solubilized receptors retain a high affinity (K_a = 1.85 ± 0.3 nM^?1), comparable to the affinity measured in membrane particles (K_a = 3.25 ± 0.7 nM^?1), and a preserved specificity for several analogs and fragments of GnRH. At millimolar concentrations, cyclic AMP derivatives inhibit [125_I] - GnRH analog binding to both membrane particles and soluble receptors from pituitary and ovary. These results support the hypothesis that cyclic AMP may play the role of an extracellular messenger by interacting with the GnRH receptor itself.     

Characterization of a diuretic hormone receptor from the tobacco hornworm,Manduca sexta

We have characterized a diuretic hormone receptor from the tobacco hornworm, Manduca sexta. A single high affinity binding site for the 41 amino acid M. sexta diuretic hormone was found in membranes prepared from Malpighian tubules of fifth stadium larvae. The site has a Kd = 79 pM and Bmax = 3.1 pmol/mg protein. The dissociation rate constant was determined to be 0.11 min^?1 with a corresponding half-life of 6.4 min. Receptor binding of the hormone is inhibited by Ca²⁺ and Mg2⁺, while Na⁺ and K⁺ inhibit binding to a lesser extent. Truncated diuretic hormone analogs in which up to 20 amino acids were removed from the N-terminus maintain high affinity for the receptor. A diuretic hormone from Locusta migratoria which has 43% sequence identity with the M. sexta diuretic hormone also possesses a high affinity for the receptor. Conformational analysis of the M. sexta diuretic hormone indicates the core region of the peptide assumes a helical conformation, which may have implications in the binding of the peptide to the receptor. © 1993 Wiley-Liss. Inc.     

High affinity binding sites for somatostatin to rat pituitary

Binding sites for somatostatin (SS) are described in rat pituitary membranes using either [¹²⁵I-Tyr¹¹]-SS-14 or [Leu⁸, D-Trp²², ¹²⁵I-Tyr²⁵]-SS-28 as radioligands; in each case saturable and high affinity binding sites with K_D's for SS of 1.09 and 0.95 nM respectively have been characterized. The binding capacity is 100 f mols/mg protein. The potencies of various SS analogs measured in the radioreceptor assay are in agreement with the potencies in a bioassay measuring inhibition of growth hormone release; in particular, SS-28 is slightly less potent than SS-14. A comparison of these data with those describing SS binding in brain and pancreas suggests that some pharmacological differences may exist between pituitary, brain and pancreas binding sites for SS.     

Synthesis and biological activity of [D-Trp6] chicken luteinizing hormone-releasing hormone

An agonist of chicken hypothalamic luteinizing hormone-releasing hormone (cLH-RH). [D-Trp⁶] cLH-RH, was synthesized and tested for luteinizing hormone (LH)-releasing activity using dispersed chicken anterior pituitary cells, as well as for binding to rat anterior pituitary membrane receptors. cLH-RH and mammalian LH-RH (mLH-RH) gave identical dose-response curves in stimulating chicken LH release ($\text{ED}{}_{50}\text{=1.6}$ and $\text{1.8×10}{}^{\mathrm{?9}}\text{M}$ respectively) and similar estimates of potency. The [D-Trp⁶] analogs of cLH-RH and mLH-RH stimulated LH release at lower doses ($\text{ED}{}_{50}\text{=7.0}$ and $\text{～7.0×10}{}^{\mathrm{?11}}\text{M}$ respectively) and were approximately 20-fold more potent. In contrast to the activity in the chicken bioassay, cLH-RH bound to rat anterior pituitary membrane receptors with a much lower affinity than did mLH-RH and had a relative potency of 2%. [D-Trp⁶] cLH-RH was approximately 100-fold more potent than cLH-RH in the rat receptor assay while [D-Trp⁶] mLH-RH was 28-fold more active than mLH-RH. These data demonstrate that substitution of Gly⁶ of LH-RH with D-Trp enhances the LH release from chicken pituitary cells to a similar extent to that observed in mammals, and indicate that the approaches used to produce active LH-RH analogs in mammals are likely to be applicable to birds.     

Binding of gonadotropin-releasing hormone (Gn-RH) to pituitary membranes in male rats

The binding of gonadotropin-releasing hormone (Gn-RH) to membranes of male rats pituitary cells was studied. It appeared that Gn-RH was bound to 3 classes of sites and that the concentration in high affinity binding sites was very low. Moreover there was negatively cooperative interaction among these high affinity binding sites leading to a rapid decrease in binding affinity and consequently unmeasurable displacement of labeled hormone with large amounts of radio-inert ligand. These facts are inconsistent with the possible estimation of Gn-RH by radioreceptor assay.     

Inferences about the conformation of somatostatin at a biologic receptor based on NMR studies

Examination of two diastereomeric analogs of somatostatin differing in stereochemistry at the tryptophan residue has revealed a high field resonance in the -Trp isomer which is assigned to the γ-methylene of Lys⁹. The extent of correlation of this shift with biologic activity for a series of analogs of somatostatin is discussed. From comparison of close analogs, it is suggested that the biologically active conformation of somatostatin at the receptor controlling insulin release is not the major conformation of this hormone in solution. It is suggested that the conformation of somatostatin at this receptor resembles more closely the solution conformation of analogs having tryptophan in the -configuration. This latter conformation places the Trp⁸-Lys⁹ side chains in close proximity, thus shifting the γ-methylene protons of Lys⁹ upfield.     

The mobile receptor hypothesis and "cooperativity" of hormone binding. Application to insulin.

The mobile receptor hypothesis has been proposed to describe the process by which hormone receptor binding initiates a biological response; it states that receptors, which can diffuse independently in the plane of the membrane, reversibly associate with effectors to regulate their activity. The affinity for effector is greater when the receptor is occupied by hormone. A mathematical expression of the mobile receptor hypothesis is used to show that: (1) The predicted kinetics of hormone receptor binding may be indistinguishable from "negative cooperativity." (2) Receptor occupancy and biological response may be coupled in a non-linear fashion. By choosing specific parameters, most of the existing data on insulin binding and biological responses can be explained in terms of the mobile receptor hypothesis. Thus, the following are easily explained: (1) A single homogeneous receptor may appear kinetically to be composed of two classes (of high and low affinity) of receptors. (2) Occupancy of the apparent class of high affinity receptors is related linearly to the biological response. (3) The same receptor in different tissues may appear to have different affinity. (4) The binding of different biologically active insulin analogues may exhibit different degrees of "cooperativity." These considerations may also be pertinent to interpretations of other hormone-receptor systems and of various ligand-macromolecule interactions.     

The mobile receptor hypothesis and “cooperativity” of hormone binding. Application to insulin

The mobile receptor hypothesis has been proposed to describe the process by which hormone receptor binding initiates a biological response; it states that receptors, which can diffuse independently in the plane of the membrane, reversibly associate with effectors to regulate their activity. The affinity for effector is greater when the receptor is occupied by hormone.A mathematical expression of the mobile receptor hypothesis is used to show that: (1) The predicted kinetics of hormone receptor binding may be indistinguishable from “negative cooperativity”. (2) Receptor occupancy and biological response may be coupled in a non-linear fashion.By choosing specific parameters, most of the existing data on insulin binding and biological responses can be explained in terms of the mobile receptor hypothesis. Thus, the following are easily explained: (1) A single homogeneous receptor may appear kinetically to be composed of two classes (of high and low affinity) of receptors. (2) Occupancy of the apparent class of high affinity receptors is related linearly to the biological response. (3) The same receptor in different tissues may appear to have different affinity. (4) The binding of different biologically active insulin analogues may exhibit different degrees of “cooperatively.” These considerations may also be pertinent to intepretations of other hormone-receptor systems and of various ligand-macromolecule interactions.     

Juvenile hormone binding components of locust fat body

Juvenile hormone (JH) binding components from the fat body of the African migratory locust were analyzed in a search for a potential nuclear JH receptor. Biosynthetically prepared 10R[³H]JH III gave a high proportion of specific binding to isolated nuclei and extracted proteins; data obtained with the JH analogs, [³H]methoprene and [³H]pyriproxyfen, on the other hand, were obscured by abundant non-specific binding. The vast majority of the high affinity JH III binding activity present in cytosolic and nuclear extracts was due to a high molecular weight JH binding protein (JHBP) which has previously been identified in locust hemolymph. This protein has several chromatographic forms which interfered in the search for a nuclear JH receptor. When specific antiserum was used to remove JHBP from nuclear extracts, a novel JH binding activity (NBP) was detected. NBP could be separated from JHBP by precipitation with ammonium sulfate. NBP displayed a high affinity for JH III (Kd = 0.25 nM) and JH I and JH II competed strongly for JH III binding, whereas methoprene and pyriproxyfen showed apparent competition when present in 1,000-fold excess. NBP was present in nuclear extracts at approximately 25,000 sites per cell; levels were similar in male and female locusts and were not greatly affected by the presence or absence of JH. The characteristics of NPB make it a strong candidate for a nuclear JH receptor. © 1995 Wiley-Liss, Inc.     

Structure-conformation-activity studies of glucagon and semi-synthetic glucagon analogs

Summary Examination of glucagon structure-activity relationships and their use for the development of glucagon antagonists (inhibitors) have been hampered until recently by the lack of high purity of semisynthetic glucagon analogs and inadequate study of full dose-response curves for these analogs in sensitive bioassay systems. Recently a number of highly purified glucagon fragments and semi-synthetic analogs have been prepared and their full dose-response activities examined over a wide concentration range using the hepatic membrane adenylate cyclase assay, the hepatic membrane receptor binding assay, and glycogenolytic activity in isolated rat hepatocytes. The results of these studies have enabled us to identify and dissociate the structural (and in some cases conformational) features of glucagon important for binding from those most responsible for biological activity (transduction). Key findings in these studies were the observation that: (1) the C-terminal region of glucagon is primarily of importance for hormone binding to receptors; (2) glucagon_1–21 and glucagon_1–6 have low potency, but are essentially fully active glucagon derivatives; and (3) highly purified glucagon_2–29 ([1-des-histidine]-glucagon), [1-N-carbamoylhistidine]-glucagon and [1-N-carbamoylhistidine, 12-N-carbamoyllysine]-glucagon are all partial agonists.These and other findings led us to synthesize several semisynthetic analogs of glucagon which were found to possess no intrinsic biological activity in the hepatic adenylate cyclase assay system, but which could block the effect of glucagon (competitive inhibitors) in activating adenylate cyclase in this system. Two of these highly purified analogs [1-des-histidine] [2-N-trinitrophenylserine, 12-homoarginine]-glucagon and [1-N-trinitrophenylhistidine, 12-homoarginine]-glucagon were quite potent glucagon antagonists (inhibitors) with pA₂ values of 7.41 and 8.16 respectively. The latter compound has also been demonstrated to decrease dramatically blood glucose levels of diabetic animals in vivo. These results demonstrate that glucagon is a major contributor to the hyperglycemia of diabetic animals.Examination of the known and calculated conformational properties of glucagon provide insight into the structural and conformational properties of glucagon and its analogs most responsible for its biological activity. Consideration of these features and the mechanism of glucagon action at the membrane receptor level provide a framework for further developing glucagon analogs for theoretical and therapeutic applications.     

Characterization of prolactin-releasing peptide: binding, signaling and hormone secretion in rodent pituitary cell lines endogenously expressing its receptor

The recently discovered prolactin-releasing peptide (PrRP) binds to the PrRP receptor and is involved in endocrine regulation and energy metabolism. However, its main physiological role is currently unknown. Two biologically active isoforms of PrRP exist: the 31 (PrRP31) and the 20 (PrRP20) amino acid forms, which both contain a C-terminal Phe amide sequence. In the present study, the PrRP receptor was immunodetected in three rodent tumor pituitary cell lines: GH3, AtT20 and RC-4B/C cells. The saturation binding of radioiodinated PrRP31 to intact cells demonstrated a K_d in the 10⁻⁹ M range and a B_max in the range of tens of thousands binding sites per cell. For binding to RC-4B/C cells, both PrRP31 and PrRP20 competed with ¹²⁵I-PrRP31 with a similar K_i. The C-terminal analog PrRP13 showed lower binding potency compared to PrRP31 and PrRP20. All PrRP analogs increased the phosphorylation of MAPK/ERK1/2 (mitogen-activated phosphorylase/extracellular-regulated kinase) and CREB (cAMP response element-binding protein) in RC-4B/C cells. Additionally, prolactin release was induced by the PrRP analogs in a dose-dependent manner in RC-4B/C cells. Finally, food intake after intracerebroventricular administration of PrRP analogs in fasted mice was followed. Both PrRP31 and PrRP20 decreased food intake, but PrRP13 did not show significant effect. Studies on pituitary cell lines expressing the PrRP receptor are more physiologically relevant than those on cells transfected with the receptor. This cell type can be used as a model system for pharmacological studies searching for PrRP antagonists and stable effective PrRP agonists, as these drugs may have potential as anti-obesity agents.     

Design, synthesis, and evaluation of fused heterocyclic analogs of SCH 58261 as adenosine A2A receptor antagonists

SCH 58261 is a reported adenosine A_2A receptor antagonist which is active in rat in vivo models of Parkinson’s Disease upon ip administration. However, it has poor selectivity versus the A₁ receptor and does not demonstrate oral activity. Quinoline analogs have improved upon the selectivity and pharmacokinetics of SCH 58261, but were difficult to handle due to poor aqueous solubility. We report the design and synthesis of fused heterocyclic analogs of SCH 58261 with aqueous solubility as well as improved A_2A receptor binding selectivity and pharmacokinetic properties. In particular, the tetrahydronaphthyridine 4s has excellent A_2A receptor in vitro binding affinity and selectivity, is active orally in a rat in vivo model of Parkinson’s Disease, and has aqueous solubility of 100 μM at physiological pH.     

Analogs of thyrotropin-releasing hormone (TRH): Receptor affinities in brains,spinal cords,and pituitaries of different species

[³H](3-Me-His²) thyrotropin-releasing hormone ([³H]MeTRH) bound to TRH receptors in rodent, rabbit and dog brain and spinal cord (SC), and in rat, sheep, bovine and dog anterior pituitary (PIT) glands, with high affinity (dissociation constants, K_ds=5–9 nM; n=3–4) but to different densities of these sites (B _max range 6–145 fmol/mg protein) (rabbit SC>sheep PITG.pig brain>dog brain>rat brain>bovine and dog PIT). Various TRH analogs competitively inhibited [³H]MeTRH binding in these tissues with a similar rank order of potency: MeTRH>TRH> CG3703RX77368MK-771>TRH Glycinamide>Glu¹-TRHCG3509NVal²-TRH>>>TRH free acid>>>and cyclo-His-Pro, indicating a pharmacological similarity of CNS and pituitary TRH receptors. While most TRH analogs displaced [³H]MeTRH binding with a similar potency in the different species, TRH exhibited a 2-fold lower affinity in the rat and G.pig brain than in other tissues of other species. Similarly, CG3703 was 2.4–4.5 times more active in the rabbit brain than in the rodent and dog brain, and also more potent in the rabbit brain as compared to the sheep PIT. However, MK-771 and RX77368 had a similar affinity for the brain TRH receptors in the different species but RX77368 was 2-fold more active in the SC preparations and 3–4-fold less active in the sheep PIT when compared to the brain homogenates. RX 77368 exhibited the highest affinity for the dog PIT TRH receptor. In contrast, MK-771 showed a similar affinity for the brain, SC and PIT TRH receptor apart from in the rat PIT where it had the highest affinity. Similarly, TRH glycinamide was more active in the dog brain than rodent and rabbit brain. These data suggest that while the rank order of potency of TRH analogs is similar in the species examined, certain analogs appear to be more potent in certain tissues of some species than in others. In addition, the current results have shown that CG3703 is almost equipotent with RX77368 and MK-771 in most species but is substantially more active than its related analog, CG3509 in the brain, SC and PIT. Taken together, these observations may have some relevance to the future clinical applications of these metabolically stabilized TRH analogs.