Kinetic analysis of thyrotropin-releasing hormone binding in the central nervous system: evidence for receptor desensitization

Neuroscience Letters, 79 (1987) 157-162

157

Elsevier Scientific Publishers Ireland Ltd. NSL 04730

Kinetic analysis of thyrotropin-releasing hormone binding in the central nervous system: evidence for receptor desensitization E.F. Hawkins and W.K. Engel u s c Neuromuscular Center, Los Angeles, CA 90017 (U. S.A. )

(Received 28 January 1987; Revised version received 30 March 1987; Accepted 7 April 1987) Key words: Thyrotropin-releasing hormone (TRH); Motor neuron; Central nervous system TRH-recep-

tor; Kinetics; Refractoriness; Bacitracin; Guanine nucleotide; Rat To investigate the mechanism(s) of experimentally and clinically observed refractoriness of spinal lower motor neurons (LMNs) to the excitatory effects of high-dose TRH, we examined the kinetics of dissociation of [3H]TRH from its CNS-receptor. At 23°C, the receptor was rapidly (40 min) and completely converted from a form with fast dissociation kinetics (complex I; tt/2 20-30 min) to one from which the peptide dissociated much more slowly (complex II; tt/2> 120 rain). This conversion required the presence of added agonist ([3H]TRH) and was not prevented by the GTP-analog Gpp(NH)p. We suggest that complexes I and II may respectively represent active and inactive (desensitized) forms of the TRH-receptor and that TRH-induced I to II conversion of the receptor is responsible for refractoriness of LMNs to the drug.

We have recently hypothesized that the rapidly developing refractoriness (tachyphylaxis) o f spinal lower m o t o r neurons ( L M N s ) to the excitatory effects o f highdose T R H might result f r o m agonist-dependent T R H - r e c e p t o r desensitization [5]. We now report d e m o n s t r a t i o n o f a rapid temperature- and ligand-dependent alteration in the rate o f dissociation o f T R H f r o m its C N S receptor (fast to slow) and postulate that these kinetically distinct forms respectively represent active and desensitized states o f a single T R H - r e c e p t o r . Male S p r a g u e - D a w l e y rats (280-300 g) were the source o f brain tissue (pyriform c o r t e x / a m y g d a l a (PC/A)) used in these studies. Preparation o f crude m e m b r a n e suspensions was performed as previously described [5, 6]. R e c e p t o r assays with [3H]TRH (107.8 Ci/mmol; D u P o n t - N E N , Boston, M A ) were conducted in P E I B A buffer (20 m M potassium phosphate, p H 7.4 at 4°C, containing 0.1% bovine serum albumin and selected peptidase inhibitors (1 m M E G T A , 0.5 m M iodoacetamide and 177/~M bacitracin)) [6]. Correspondence: E.F. Hawkins. Present address: Lundon Software Inc., P.O. Box 21820, Cleveland, OH

44121, U.S.A. 0304-3940/87/$ 03.50 O 1987 Elsevier Scientific Publishers Ireland Ltd.

158

Bacitracin was included to limit the formation of deamidated products from TRH [13]. Although we had previously demonstrated that this compound did not prevent receptor binding of [3H]TRH at 0°C [6], others noted a reduction in bound [3H]methyl-His2-TRH([3H]MeTRH) in the presence of micromolar concentrations of bacitracin at 37°C [1, 10]. Since our kinetic experiments included incubations at 23°C (see below), we first examined the effects, if any, of bacitracin on [3H]TRH catabolism and receptor binding at this temperature. PC/A membranes were incubated at 23°C in buffer_bacitracin (177 #M) with [3H]TRH (10 nM) in the absence (total binding) and presence (non-saturable binding) of 4 #M unlabeled TRH, from which specific binding was determined by difference. Incubations were terminated by the addition of 2 ml of ice-cold buffer, followed by vacuum filtration on glass fiber filters (Schleicher and Schuell, Keene, NH) at 2°C with 3 x 2 ml ice-cold buffer washes of tubes and filters. Filters were transferred to scintillation vials and extracted in scintillation fluid (ScintiVerse II; Fisher Scientific) for 36 h prior to counting with dpm correction. Protein concentrations were determined by the method of Lowry et al. [9], using bovine serum albumin as standard. Samples for thin-layer chromatography (TLC) were rapidly centrifuged at 2°C, the pellets were surface-rinsed twice with icecold buffer and extracted with 50/tl methanol for 30 min at 2°C. Extracts were centrifuged and stored under liquid nitrogen until analyzed. TLC was performed as previously described [6]. Receptor occupancy by [3H]TRH was not reduced by bacitracin at 23°C. Binding after 40 min with or without bacitracin was identical (16 + 3 and 16 ___4 fmol/mg prot. respectively; means _ S.D. of 3 experiments). Nevertheless, bacitracin did effectively limit catabolism of [3H]TRH seen in its absence (Fig. 1) and was thus retained in the assay buffer. Our kinetic analyses of [3H]TRH dissociation from the CNS TRH-receptor were

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Fig. I. Effect of bacitracin on [3H]TRH catabolism by PC/A membranes: analysis by TLC. Membranes were incubated_+_bacitracin with [3H]TRH, as described in the text. Methanol extracts (10 #1) of membranes were applied to Whatman LHP-KD silica-gel plates and chromatographed in the system chloroform: methanol:ammonium hydroxide (28%) (5:3:1). 3H-labeled standards included TRH, cyclo (His-Pro) (cliP), proline and histidine and unlabeled standards were TRH, cliP, TRH-OH and His-Pro. Unlabeled peptide standards were visualized with diazotized sulfanilic acid.

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Fig. 2. Effect of the time and temperature of labeling on subsequent [3H]TRH dissociation from its CNS receptor (PC/A). Details of the experimental protocol are provided in the text. Curves are from a representative experiment (3 performed for each condition). Shown is the computed best-fit to the data points (-) together with the rapidly dissociating (I,...) and slowly dissociating (II, - - -) components, where separately resolved.

based on an earlier report [7] indicating that the temperature of labeling of pituitary TRH-receptors with [3H]TRH strikingly affected dissociation of the peptide from the receptor complex. We compared dissociation of [3H]TRH following initial receptor labeling at 0 or 23°C. Parallel incubations were performed with [3H]TRH (I0 nM) in the absence (total binding) or presence (non-saturable binding) of the unlabeled peptide (5/zM). In pilot experiments (not shown), we found that saturable binding of [3H]TRH was complete by 1 h at 0°C and used these conditions for subsequent 0°C labeling. Similar levels of saturable binding to those measured at 0°C/1 h were reached by 20 or 40 min at 23°C (Fig. 2), indicating no significant loss of receptor sites at this temperature. Following labeling, the dissociation phase of all experiments was performed at 0°C. The temperature of the 23°C incubates was rapidly (5 min) reduced to below I°C (ice-water bath). Incubates were transferred to ice and unlabeled TRH was added to the total binding tube (final concn.= 10 pM). At various times, triplicate 300-/tl aliquots of the incubated were vacuum filtered and bound radioactivity measured as described earlier. Non-linear least squares regression analysis of the dissociation data was performed by an exponential curve analysis program (Lundon Software, Cleveland, OH). Statistical evaluation of the computed curve-fits included: (a) standard errors of the parameter estimates; (b) residual sum of squares error of the fitted curve; (c) a test to detect non-randomness in the data set; (d) an F-test for model comparison (1 vs 2 site non-linear fit). Results from 3 separate experiments for each labeling condition are summarized in Table I and computer-generated curve fits from individual experiments are shown in Fig. 2. We observed a rapid (40 min to completion), temperature-dependent conversion of the [3H]TRH-receptor complex from a form with fast dissociation kinetics (complex I; t]/2 of 20-30 min) to a form (complex II) from which [3H]TRH dissociated much more slowly (tl/2 greater than 120 min). When the labeling phase of the experiments was performed at either 0°C for 1 h or 23°C for 20 min, both components

160 TABLE I EFFECT OF LABELING CONDITIONS ON DISSOCIATION OF [3H]TRH FROM ITS CNSRECEPTOR Values expressed as mean ± S.D. of 3 experiments (*2 experiments). Gpp(NH)p, guanyl-5'-yl imidodiphosphate. No rapidly dissociating component was resolved following labelling at 23°C/40 min. Component

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2.7+0.3 25 ±4.0 22 ± l . 0

3.6±0.4 19 ±2.0 I0 +_3.2

Slow Ko~(min l× 10 3) t~/2(min) Binding (frnol/mg prot.)

4.3± 0.9 165 ±36 6 + 3.0

5.3+ 0.8 133 -I-21 16 ± 1.4

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5.5+0.2 126 +4.0 18 -I-2.0

were clearly resolved (Fig. 2A, B). By 40 min at 23°C, conversion to complex II was complete (Fig. 2C). Irrespective of labeling temperature, the total amount of receptor sites measured at time 0 (start of dissociation phase) was similar, indicating that loss of complex I during labeling at 23°C could not be explained by receptor degradation. We next determined whether added agonist ([3H]TRH) was required to facilitate the conversion of complex I to complex II at 23°C. In two separate experiments, PC/ A membranes were incubated without added ligand at 23°C for 40 min (conditions under which formation of complex II was complete in the presence of [3H]TRH (Fig. 2C)). The receptor was then labeled with [3H]TRH (10 nM)+__unlabeled TRH (5/~M) for 1 h at 0°C and peptide dissociation was monitored at 0°C as previously described. Results were similar to those obtained when the receptor was labeled with [3H]TRH at 0°C without the elevated-temperature preincubation step. Complexes I (tim, 30 + 7 min; 19+2 fmol/mg prot.) and II (tin, 161 + 2 min; 9 + 2 fmol/mg prot.) were both resolved with the former predominating (69 + 3% of total measured receptor sites). Importantly, these preincubation experiments demonstrated that added [3H]TRH was required for the previously observed complete complex I to II transformation by 40 min at 23°C (Fig. 2C); elevated temperature alone was insufficient to cause this alteration in the kinetics of the receptor complex. Possibly, residual complex II (comprising 30% or less of total labeled receptor) found, (a) in the preincubation experiments, and (b) following 0°C labeling without preincubation (Table I), may reflect receptor transformation by bound, endogenous TRH present in the tissue and subsequent exchange of this for [3H]TRH during labeling, without prior reconversion of the receptor to complex I.

161 Biphasic dissociation kinetics for the CNS TRH-receptor have previously been reported by others following receptor labeling with [3H]MeTRH [10, 11]. However, to our knowledge, our data represent the first demonstration o f agonist-dependent complete conversion of the CNS TRH-receptor from a rapidly to a slowly dissociating form upon binding o f [3H]TRH. Rather than favoring receptor heterogeneity, we interpret these findings as representing kinetically distinct states of a single T R H receptor in rat CNS, a view supported by our previous studies including: (a) identification of only a single high-affinity site by saturation binding experiments with up to 100 nM [3H]TRH [6] and (b) monophasic competition curves with unlabeled T R H and analogs [4, 6]. Both pituitary [7] and CNS (this study) TRH-receptors are similarly converted from rapidly to slowly dissociating forms when labeled with [3H]TRH at elevated temperature, although the underlying mechanisms may be different. In this regard the pituitary receptor appears to be guanine nucleotide-sensitive, since micromolar concentrations of G T P or G p p ( N H ) p increased the dissociation rate o f [3H]TRH from the complex [8]. In contrast, G p p ( N H ) p at 10/tM (an effective concentration in pituitary assays [8, 12]) did not stabilize or promote the formation of the rapidly dissociating complex I during CNS receptor labeling at 23°C/40 min and subsequent dissocation at 0°C (Table II). CNS receptor binding of [3H]TRH was also unaffected by a wide range of G p p ( N H ) p concentrations (100 nM to 1 mM; data not shown) in contrast with the pituitary receptor (IC50 = 0.3/,M) [8]. Burt and Taylor [2] were also unable to demonstrate guanine nucleotide effects on the CNS TRH-receptor. One possible biochemical explanation of the rapidly developing clinical [3] and experimental [5] refractoriness o f LMNs to high-dose T R H may reside in ligandinduced conversion o f the receptor from complex I to complex II that we report here. We propose that these complexes respectively may represent active and inactive (desensitized) states of the CNS TRH-receptor. Financial support for these studies was provided by the ALS Association and the Muscular Dystrophy Association. 1 Bhargava,H.N. and Das, S., Evidencefor opiate action at the brain receptorsfor thyrotropin-releasing hormone, Brain Res., 368 (1986)262-267. 2 Burt, D.R. and Taylor, R.L., Guanine nucleotidesmodulate receptor binding for TRH in the pituitary but not in CNS, Soc. Neurosci.Abstr., 6 (1980)255. 3 Engel,W.K. and Siddique,T., Transient autorefractory state of transmitter-likeeffectof thyrotropinreleasing hormone (TRH) in amyotrophiclateral sclerosis(ALS); a new clinical phenomenon,Neurology, 34 (1984) 147. 4 Hawkins, E.F. and Engel, W.K., Analog specificityof the thyrotropin-releasinghormone receptor in the central nervous system:possibleclinical implications, Life Sci., 36 (1985)601-611. 5 Hawkins, E.F., Beydoun, S.R., Haun, C.K. and Engel, W.K., Analogs of thyrotropin-releasinghormone: hypothesesrelating receptor binding to net excitation of spinal lower motor neurons, Biochem. Biophys. Res. Commun., 138 (1986) 1184-1190. 6 Hawkins, E.F., Wade, R. and Engel, W.K., Lack of usefulness of DN-1417 for characterization of a CNS receptor for TRH, J. Neurochem.,in press. 7 Hinkle, P.M. and Kinsella, P.A., Rapid temperature-dependenttransformation of thyrotropin-releas-

162 ing hormone-receptor complex in rat pituitary tumor cells, J. Biol. Chem., 257 (1982) 5462 5470. 8 Hinkle, P.M. and Kinsella, P.A., Regulation of thyrotropin-releasing hormone binding by monovalent cations and guanyl nucleotides, J. Biol. Chem., 259 (1984) 3445-3449. 9 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 10 Sharif, N.A. and Burt, D.R., Rat brain TRH receptors: kinetics, pharmacology, distribution and ionic effects, Regul. Peptides, 7 (1983) 399~,11. 11 Simasko, S.M. and Horita, A., Characterization and distribution of 3H-(3Me-His2) thyrotropin-releasing hormone receptors in rat brain, Life Sci., 30 (1982) 1793-1799. 12 Taylor, R.L. and Burt, D.R., Guanine nucleotides modulate TRH-receptor binding in sheep anterior pituitary, Mol. Cell. Endocrinol., 21 (1981) 85-91. 13 Webster, V.A.D. and Grifliths, E.C., TRH degradation: an example of neuropeptide biotransformation. In E.C. Griffiths and G.W. Bennett (Eds.), Thyrotropin-Releasing Hormone, Raven, New York, 1983, pp. 95-102.