Baixe Dynamic Model of Gona e outras Notas de estudo em PDF para Engenharia Biológica, somente na Docsity! cer | horinons receptor function Í JOSEPH C. ZOLMAN amic model of gonadotropin- “releasing Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77550 ZOLMAN, JOSEPH C. Dynamic model of gonadotropin-releas- ing hormone receptor function. Am. J. Physiol. 248 (Regulatory Integrative Comp. Physiol. 17); R312-R319, 1985.— Function- ing gonadotropin-relessing hormone (GnRH) receptor is visu- alized as an aggregate of identical subunits (not all always functional) with the aggregate usually transformed into at least four suceessive structurally distinct receptor assemblies. Recep- tor protein, hormone molecule(s), and carrier(s) are main com- ponents of each functional subunit. During the normal life- span of the functional subunit, each carrier is responsible for delivery of a unit amount of product, per unit time, to the cell surface (ratio betwsen functional carrier and bound hormone, 1:1). Association of hormone with the receptor protein is essen- tial, not only for the initial formation of the functional subunits but also for subsequent conformational changes that are in turn essential for formation of the aggregate only, or later (in the presence of sufficiently high GnRH concentrations) for a successive formation of a family of receptor assemblies (occur- ring one at a time). The successive assemblies differ from the aggregate by being more stable and from one another by in- creasing GnRH binding affinity and apparent capacity. They resist stimulation during protracted decay (desensitization). luteinizing hormone; receptor interaction; functioning receptor; receptor protein aggregation; suecessive receptor assemblies; desensitization THE NEUROENDOCRINES, such as thyrotropin-releasing hormone (TRH) (3) and gonadotropin-releasing hor- mone (GnRH) (15), are thought to act during an effo on specific entities in the anterior pituitary plasma mem- brane that are termed receptors. Investigations with iodinated GnRH to clarify the actions of its receptor have special significance, because the iodinated GnRH has proved to be fully biologically active in organ culture of bovine anterior pituitary slices (24), and at least partially active with rat anterior pitai- tary (17), whereas TRH loses its biological activity on iodination (11). The peptide of the native amino acid sequence is a logical choice (1, 14, 19) for the recepror studies because analogs may operate through pathwe ys that are independent of normel physiological mecha- nisms—as happens with anomalous degradation (9) and cellular distribution (18). Dynamic change may be considered to be a universal biological phenomenon, which is apparent at varicus levels of organization, such as population, organis:n, organ, tissue, and cell. In the organism, for example, it may be manifest as cyclicity, periodicity, or pulsatili-y, R3I2 and it may be involved, for example, in development (as in sexual development) in feedback mechanisms or (sub- cellularly) as protein secretory processes. Dynamic change therefore is likely to be an important feature in any regulatory process. The degree to which the dynamic change is parallel among the various levels in an organism may characterize the relationship among them or possibly their depend- ence on a common factor or event. In a hormonal system the dynamic change in response may also relate to the mode of action of the triggering agent. We report here that the interaction between GnRH (the triggering agent) and its receptor may be the single most important celtular event in GnRH-stimulated luteinizing hormone (LH) release, because the response at molecular, cellular, and tissue level is parallel, A functional amplification model of the GnRfI receptor, based on evidence pre- sented mostly here, is introduced to explain how the putative receptor acts. MATERIALS Tissue culture (TC) medium 199 was purchased from Difeo (Detrcit, MI). Highly purified collagenase was from Worthington (Freeport, NJ); hyaluronidase and soybean trypsin inhibitor were (vom Sigma (St. Louis, MO); i? berglass microiilters (Whatman) were purchased through Fisher (St. Louis, MO). Synthetic GnRH was a gift from Ayerst (Mont real, Canada). Synthetic TRH was a gift of Abbott (N. Chicago, IL) and National Institutes of Health (Bethesda, MD; was the standard of LH and LH for iodination. Carrier-free 1º], as '2I-Na, was ob- tained from New England Nuclear (Boston, MA). Trans- parent nylon fihers werc from Dupont (Wilmington, DE). Water-soiuble carbodiinices and chemicals for acyl azide intermedia ivation of the solid phase were from Aldrich (Milwaukee, WI), and the decastaltic pump was purchased from Buchler instruments (Fort Lee, Nd). Bovine anterior pituitaries were obtoined fresh at an” abattoir, imimersed in the TC medium 199 at 37ºC, transported to the laboratory, and used within the next 2h. : METHODS periments, Heifers were-12-16 mo old at the ment and were observed.to have at least two ecutive estrous cycles hefore the experiment The vere palhated per rectum the'American Pliysiological Society DYNAMIC MODEL OF HORMONE RECEPTOR daily, beginni ag 4 days before ovulation, during pretreat- ment and experimental estrous. The heifers that were assigned to be treated after luteal regression were pal- pated per rectum twice daily, beginning 4 days before estrus was expected, and treatment was given within 12 h after the corpus luteum had regressed to 1.0 em in diameter. On the average this took place on day 20 of the estrous cycle. Rectal palpation revealed that no heifer ovulated before GnRH administration. Twenty-four hours before each experiment, a cannula (Vinyl Tubing, Clay Adams, New York, NY) was inserted into the left jugular vein of each animal. Synthetic GnRH in 10 ml isotonic saline was given intravenously as a bolus injection via the jugular cannula. Blood samples (10 ml) were collected at 60, 30, and 10 min before GnRH treatment, afterward at 2-min intervals for 30 min, again at 45 min, and then at 30-min intervals from 60 to 180 min. Tissue superfusion in vitro and measurement of LH. The in vitro superfusion of bovine anterior pituitary tissue slices has been tested and validated (20): the volume of the incubation chamber in the system, 0.5 ml; »w rate, 2 ml/min; effluent sample size, 1 ml; and collection interval, 0.5 min. LH was measured in every other specimen during the 10 min before, and 10 min after, the beginning of exposure of the specimen to 4 ng/ ml GnRH and in every fifth sample during the subse- quent 50 min. The tissue, harvested only from cows and only from the central area of the gland, was minced into 1X 1X 10 mm sections, carefully randomized, and preincubated with three tissue slices per chamber for 2 h before 1 h incubation with GnRH. The TC medium 199 contained 0.224% bicarbonate. The effluent fractions and serum from in vivo experi- ments were frozen on collection and stored at —2 until analysis-by a specific radioimmunoassay (RIA) that uses materials obtained from National Institutes of Health. LH release rates are given in nanogranis per milligram tissue (wet weight) per minute, compared with the NIH LH-B5 standard. Dilution curves of standard LH and experimental samples were parallel, exhibiting a high correlation. “'solated anterior pituitary cell binding. Pituitary glands ». re removed from cattle at slaughter, kept at 37 “C, and used within 2 h. The posterior pituitary was discarded, and the anterior pituitary halves were cut into rectan- gular blocks (1 X 1 x 10 mm). The blocks were immersed in an Erlenmeyer flask of freshly prepared TC medium 199 and placed in a water bath kept at 37 *C. The medium was replenished every 20 min. According to the studies of hormone secretion patterns (20), this procedure re- stores normal cell activity. After the 2-h preincubation the original medium was decanted and replaced by a 2.5-ml enzyme solution per pituitary. This solution consists of hyaluronidase (0.2%), trypsin inhibitor (0.01%), and highly purified collagenase (0.15%) in TC medium 199. The tissue was minced and the mixture shaken lightly for 15 min. Ca?* and Mg” were then removed temporarily by 2-min washes (3x) with warm (37ºC) Mg?- and Ca't-free Krebs-Ringer buffer (KRB) with 3 mM EDTA (Mg”*- and Ca?*-free A R313 KRB obtained by substituting NaCl for MgSO, and Ce Ch). Both cations were later returned in 10 ml TC medium 199. After 10 min the tissue mince was divided into several 25-ml Erlenmeyer flasks, each containing 7 ml enzyme solution. Vigorous shaking with this solution for 45 min caused a perfusate of single cells to appear in the solution. The yield can be enhanced by mild shearing with a Pasteur pipette. To remove the enzymes and cell fragments, the cell suspension was centrifuged at 50 g for 5 min; the super- natant was discarded, and the pellet was resuspended in TC medium 199. After three such washes the suspension was filtered through several layers of cheesecloth to remove small clumps of cells that aggregated as a result of'peileting. The cell concentration was then determined in a counting chamber, and the suspension was diluted in the manner prescribed lin the experimental design. “The freshly prepared suspension of cells was diluted to the required cell concentration with aerated TC medium 198. The techniques of iodination of the releasing hor- mone and binding of 'SI-GnRH to isolated anterior pituitary cells, described in detail by Zolman and Valenta (24), began with transfer of aliquots of 0.9 ml into incu- bation tubes kept in a wuter bath at 37ºC; then !2]- GnRH added in 100-ul aliquots of the same buffer was carefully mixed with the cell suspension. After incuba- tion 100-ul aliquots from each tube were transferred onto a microfilter and washed with 4 ml ice-cold buffer. Filters were allowed to dry and were counted in a y-counter. In the determination of all assays of binding, the points represent the means of quadruplicate determinations. Counts from the controls (no cells added) were sub- tracted from the experimental count. Receptor protein binding. The receptor protein was purified as described by Zolman and Valenta (25). It was homogenous on polyacrylamide gel electrophoresis, by electrofocusing, and by gel chromatography. For the studies of the purified receptor protein binding, the latter was labeled with 2! by the standard chloramine-T method (6). This was performed in 0.1 M sodium phos- phate buffer, pH 7.4, with carrier-free !>]. The iodination mixture was chromatographed on a Biogel P-150 column eluted with 0.01 M phosphate buffer, pH 7.4 The iodi- nated receptor protein was stored in 0.1 tris(hydroxy- methyl)aminomethane (Tris)-HCI buffer, pH 9.0, at 20ºC until used. A solid-phase GnRH was prepared by coupling a slightly labeled “1-GnRH to a nylon fiber of a standard size by either carbodiimide (4) or acyl azide intermediate (7) techniques. The rate of coupling was 0.5 pg decapep- tide/fiber. The binding experiments were períormed in 0.1 M Tris-HC! buffer, pH 9.0. The immobilized GnRH was incubated with !ºI-GnRH receptor protein for pre- determined time periods at 37ºC. Bound and free protein were separated by simply removing the solid phase from the solution and washing it thoroughly in an excess buffer. The solid phase was then counted in the y-counter for retained 12º] radioactivity. Statistical analysis. ANOVAs of various degrees of complexity wero used to data, Partition of the error term in split-plot ANOVA (5) was used as a prin- sc cs omg sem R316 in serum, or even smaller, is available at the cel! 7 Therefore the LH release rate after a s'ngle injection seems to be compatible with the classical definition of positive cooperativity (12). The effect of GnRH on the gonadotrepes is likely to be mediated by its receptor, which may be defined oper- ationally (in molecular terms) as preferred conforma- tional state(s) of the binding macromolecule (receptor protein) that is exclusively and directly associated with the recognized biological effect, GnRH-stimulated LH release. The affinity chromatography purified GnRH receptor protein aggregates in solution upon binding of GnRH present in physiological concentration (25, 26). The binding of labeled GnRH receptor protein to im- mobilized hormone and its kinetics, as shown here and earlier (23, 26), are consistent with an interpretation (GnRH receptor protein aggregation) of change in the conformation of the binding protein on binding of the hormone. In addition, to explain the parallelism of GnRH responses at molecular, cellular, and tissue levels that our data demonstrate, it is necessary to postulate that every binding component of the receptor protein has an adjacent carrier or mechanism capable of prompt delivery of a discrete amount of GnRH to the gonado- trope surface. When “turned on,” this carrier would mediate the response. Such an arrangement would pro- vide for the manifestation of structural changes in recep- tor to be manifest biologically, (i.e. in vitro). These structural changes (conformational states) would be characterized further by thé affinity and capacity of the hormone for binding. The degree of biological effect would be determined exclusively by the hormone's oc- cupancy of receptor binding sites, within the overall- constraints specified in the model. MODEL The biochemical and physioiogical data derived in this study can be explained by a relatively simple dynamic model, a functional amplification of the receptor fune- tion. Let us assumé that a carier is adjacent to every binding component of the receptor protein and, as long as the hormone molecule remains bouná, the carrier continues to deliver a discrete amount of product, per unit time, to the cell surface (exocytosis). Initially, ón binding of a small amount of hormore, the functional subunits, each consisting of carriers, re- ceptor protein, and hormone molecules, simultaneously begin exocytosis and acquire a tendency to aggregate (ascending leg of the peak, Fig. 5). Thus an aggregate of functional subunits is formec by specific aggregaticn, whereas the functional subunits continue exocytosis.” Formation of such an aggregate is instrumental later in the receptor-mediated positively cooperative binding and biological response. The aggregate is relatively unstab'e; ! Aggregation is used to mean a simple noncovalent binding (asso- ciation) as originally introduced (22, 25) and subsequently confirmed by independent means, our studies (28), and others (e.g., Ref. 10). ft does not imply any degree of cross-link, as is sometimes invoked in related literature (8, 16) because of a precipitin-type reaction (13). 3. C. ZOLMAN Assemblyll pp Assembly 1 Ed Aggregotod FUNCTION ——+ TIME-minutes FIG. 5. Schematic representation of selected dynamic structural changes of gonadotropin-releasing hormone (GnRH) receptor as they relate to hypothetical function, according to dynamic model of GnRH receptor function. Only initial transition (aggregation) from ground state has been experimentally confirmed with GnRH receptor protein in solution (28). Remaining conformational rearrangements in receptor have been postulated, but configuration is hypothetical. Self-explana- tory, for details see text. the hormone dissociates with a fast dissociation constant, and the rate of exocytosis also decreases significantly (descending leg of the peak, Fig. 5). In the presence of sufficient concentrations of the hormone in the surrounding environment, however, the original aggregate can undergo restructuring into an as- sembly, designiated here for convenience to be of the first order. The assembly binds the hormone molecules with a dissociation constant that is slower (higher) than that of the initial aggregate (first plateau, Fig. 5). After a lag period (flat portion of the first plateau, Fig. 5), the assemblies of the first order cach pick up one or more molecules of the hormone from the surrounding environ- ment (second plateau, Fig. 5). Thus the assembly for- mation (which is, alone, mediated by the bound hormone) facilitates, or creates, binding to a new site for a subse- quent molecule to be bound with greater affinity, satis- fying the main conditions of positive cooperativity. After yet another lag period, the second-order assem- blies formed from the first-order assemblies may restruc- ture again, and the process for making sites available for new hormone molecules is repeated (full cycle, Fig. 6). Because the bound hormone molecule converts a non- functioning carrier (“turns the carrier on”), through the binding component, into a functioning one, the increase in the rate of release of the product (rate of exocytosis) reflects proportionately the magnitude of the binding of hormone to the receptor. DYNAMIC MODEL OF HORMONE RECEPTOR R317 rrreevesme AGO REONTION é RECEPTOR PROTEIN & ção Mansion” CARRIER mem nesmUCIURINO * NoRMONE meme DISAGGREGATON & FUNCIONAL SUBUNIT ——— orar Ze PAIHWAY DIRECHON | AGGREGATED Õ . ASSEMBLY IL ASSEMBLY DE ss [mts FINAL Fay VD ASSEMBLY 14 sa | e LOS riG. 6. Dynamic model of gonadotropin-re- N leasing hormone (GnRH) receptor function. g a GnRH binding and its hypotheticel structural “ o N and functional correlates. “Twô-dimensional a plane coordinate system selected for easier visu- y % alization. Receptor is functioning (rate deter- q mined by number of functional subunits) except o. ê % for ground state, transitional state, and final ASSEMBLY Ê | %» stages of assembly decay. Assembly formation is DECAY STATES É & contingent or availability ot GnRH in concentra- 8 y tions to satisfy affinity requirements of positively / & é cooperative receptor at any particular time. As- . & Es sembly decay states correspond functionally to ao &3 desensitization. As drawn, full cycle may take ' & 12 hto con plete, aborted cycles being propor- é tionately shorter. Self-explanatory, for details see text. DECAY £ Sines É mi cogonte? Ega Ê a For the cycle of further restructuring (increase in function) to stop (“aborted ”: assembly decay < 25, Fig. 6), it would be su molecules be present in the solution or the cellular en- vironment at the time of the formation of an assembly of a particular order. This is because all sites available for binding, in any assembly, must be occupied for the (functional) amplification cycle to continue. Similarly, when only a low hormone concentration is present in the environment at the time of formation of the assembly, and there is an insufficient concentration to satisfy the affinity requirements of the assembly of a particular order, the (functional) amplification cycle is interrupted, and the binding maximum as well as a peak in product release rate have been reached. The product release rate then begins to diminish in accordence with the rate of decay of the receptor assembly. Furthermore, because the receptor assembly es pre- cludes any other type of receptor restructyring.and thus, n prod se rate, what ensues, in funetional terms, is desensitization, defined as à grad- * defined in thi ual development of a temporary lack of responsiveness. Essentially the gonadotropes cease to respond to (free) GnRH stimulus during desensitization, but because the receptor assembly decay and the accompanying hormone dissuciation are both protracted phenomena, the product release continues, although in an ever-decreasing rate. Refractoriness, the temporary lack of response, is the state when the product release rate has bottomed out (decreased to base line) and the receptor has decayed into its elementary components (carrier, hormone, recep- tor protein) or their metabolites, but neither responds to stimulus (by increased product release rate and hormone binding, respect vely). Likewise subsensitivity (submax- imal sensitivity) describes the receptor's failure (struc- tural or temporal) to reach the expected or final state (receptor assembly) of the functional amplification series that is ordinarily characterized by a definite or maximal proguct release rate, respectively. For completion, another familiar term, thrs shold, is nodel as the lowest cone hormone to trigger response (i.e., to cause a change in sima à a EC da o cs R318 GnRH binding and rate of product release). Obviously- the absolute threshold has a unique value; it is the low concentration oí the hormone that increases the binding (and-raises the minimum increase in the detectable rate of product release over base line). Several “apparent” thresholds would be expected, however, depending on the functional state of the receptor, as characterized by the presence of a particular assembly. Ascending from the assembly of the first order, the threshold vould decrease (affinity increased); binding capacity woulc incrense, and product release rate would increase with the occurrence of the receptor assembly of each higher order, except the final one, because of positive cooperativity of the recep- tor. Many physiological ramifications of this model are apparent, and others are of only theoretical interest at the present time. The dynamic supramolecular structure, composed of receptor ground state, aggregate(s), assem- blies, and decay state(s), appears more as a discriminant that is recognized biologically than a conventional recep- tor (receptive substance). However, one of its aspects, reversibility of the initial hormone receptor (plus carrier) interaction, which that leads to subunit aggregation and an apparent subsequent hormone dissociation, is now of keen practical interest. It explains why intermittent time-spaced injections of low doses of GnRH in vivo (pulses), or a short-term exposure to even higher concen- trations in vitro, do not lead to desensitization, whereas the opposite (high doses, constant exposure) does. The theoretical basis for intermittent regimens (but not con- REFERENCES - BAUMANN, R., AND H. KUHL. Interaction of [“SI)LH-RH and other oligopeptides with plasma membranes of rat anterior pitui- taries. Acta Endocrinol. 92: 228-241, 1979. BEnNETT, H. P. J., AND C, MCMARTIN. Peptide hormones and their analogues: distribution, clearance from the circulation, and inactivation in vivo. Pharmacol. Rev, 30: 247-292, 1979. . BURGUS, R., T. F. DUNN, D. DESIDERIO, AND R. GUILLEMIN. Structure moléculaire du facteur hypothalamique hypophisiotrope TRF d'origine ovine: mise en évidence par spectrometrie de masse de la sequence PCA-His-Pro-NHs. CR Acad. Sei, Ser. D. 269: 1870- 1878, 1969, EDELMAN, G. M., AND U. RUTISHAUSER, Specific fractionation and manipulation of cells with chemically derivatized fibers and surfaces. Methods Enzymol. 34: 195-225, 1974. Gis, J. L. Design and Analysis of Experiments in the Animal and Medical Sciences. Ames, IA: Jowa State Univ. Press, 1978, vol. 1, p. 176-207. HunTER, W. M., AND F. C. GREENIWOOD. Preparation of iodine- 131 labelied human growth hormone of high specific activity. Nature London 194: 495-496, 1962. INMAN, J. K., AND H. M. DINTZIS. The derivatization of crose- linked polyacrylamide beads. Controlle.l introduction of functionel groups for preparation of special-pu-pose, biochemical absorbents. Biochemistry 8: 4074-4082, 1969. KAHN, C. R., K. L. Biro, D. B. JAKRETT, AND J. S. FUER. Direct demonstration that receptor crosslinking or aggregation is impor- tant in insulin action. Proe. Natl. Acad. Sci USA 75: 4209-4213, 1978. KOCH, Y., T. BARAM, E. HAZUM, AND M. FRIDKIN. Resistance to enzymatic degradation of LH-RH analogues possessing increased biological activity. Biochem. Biophys. Res. Commun. 74: 488-496, 1977. 10. LimpBirD, L. J., AND R. J. LEPKOW-TZ. Agonist-induced increase in apparent $-adrenergic receptor size. Proc. Natl. Acad. Sci. US/ 75: 228-209, 1978. e 2 a se J. C. ZOLMAN' > stant éxposure) to be effective is that receptor restiue- tiring from: the aggregate to various bli = viated (at the level of the first assembiy), and thus ail receptor decay states are not involved (desensitization, see above). Conversely, to effect a long-term desensitization, such as for the purposes of contraception or cancer therapy, one would need to prevent the formation of the aggregate as well as of the various assemblies. Arrest of the receptor decay in any of its states, or prevention of the formation of the ground state, would accomplish essentially the same task. In summary, in systems such as the one described here, the biological response to hormone (such as exocytosis in the target cell under both in vivo and in vitro condi- tions) depends not only on the concentration of hormone that causes the original response but also on the length of exposure to this concentration—or an even lower one. These are the equilibrium and kinetic features and re- quirements of an apparent, positively cooperative (19), response. Such an interpretation is also compatible with physiologically observed phenomena such as threshold, subsensitivity, desensitization, refractoriness, and the steep sigmoid dose-response curves and concentration- dependent binding curves. The dynamic model of GnRH receptor function presented here is an attempt at a mechanistic explanation of receptor role in control of the rate of GnRH-stimulated LH release. | Received 20 December 1982; accepted in final form 1 October 1984. - MARTINO, E., H. SEO, AND S. RerpTOPF. Loss of bioreactivity and preservation of immunoreactivity of iodothyrotropin-releasing hor- mone, Endocrinology 103: 246-253, 1978. 12. NEgr, K. E. Cooperativity in enzyme function: equilibrium and kinetic aspects. Methods Enzymol. 64: 139-192, 1980. 13. PERBLSON, À. S. Receptor clustering cn a cell surface. IL. Theory = of receptor eross-linking by multivalent ligands: description by ligand states! Muth. Biosci. 53: 1-39, 1981. . Perrin, M. É, J. E. RIVIER, AND W. W. VALE. Radioligand assay for gonadotropin-releasing hormone: relative potencies of agonists and antagonists. Endocrinology 106: 1239-4296, 1980. ScriaLty, À. V., R.M. G. NAIR, T. W, REDDING, AND À. ARIMURA. Isolation of the luteinizing hormone and follicle-stimulating hor- mone-releasing hormone from porcine hypothalami. d. Biol. Chem. 246: 7230-7236, 1971. Scncurer, Y., K. J. CHANG, S. JACOBS, AND P. CUATRECASAS. Modulation of binding and bioactivity of insulin by anti-insulin antibody: relation to possible role of receptor self-aggregation in hormone action. Proc. Natl Acad. Sci USA 76; 2720-2724, 1979, 17. TERADA, S., S. H. NAKAGAWA, D. C. YANG, À. LIPKOWSKI, AND G. FLouRET. Jodination of luteinizing hormone-releasing hormone. Biochemistry 19: 2572-2576, 1980. 18. WAGNER, T. O. F. Suhcellular distribution of pituitary gonadotro- pin-releasing hormone receptors (Abstract).. Acta Endocrinol. Suppl. 240: 121, 1981. 19. WAGNER, T. O. F, T. E. ADAMS, AND T. M. NeTT. GnRH interaction with anterior pituitary. L. Determination of the affínity and number of receptors for GnRH in ovine anterior pituitary. Biol. Reprod. 20: 140 -149, 1979, 16. B 20. ZOLMAN, J. C., AND E. M, CONVEY. Bovine pituitary LH and prolactin release during superfusion in vitro. Proc. Soc. Exp. Biol Med 140: 194-198, 1972. a 21. ZOLMAN, J. C., E. M. Convey, J. H. Barr, AND H. D. HAPS. Release of bovine luteiniting hormone by pufified porcine and synthetic gonadotropin releasing hormone, Proc. Soe. Exp. Biol.