Interactions of Adrenocorticotropic Hormone with Its Adrenal Receptors DEGRADATION OF ACTHI., AND ACTHmu*

Crude membranes (20,000 times g pellet) prepared from human, rat, and ovine adrenals bind 125-I-corticotropin-(1-24)-tetracosapeptide (125-I-ACTH-1-24) and degrade unbound hormone. The degradation is dependent on temperature and the concentration of membrane proteins. The degradation of 125-I-[9-tryptophan(o-nitrophenylsulfenyl)]-corticotropin-(1-24)-tetracosapeptide (125-I-NPS-ACTH-1-24) is similar to 125-I-ACTH-1-24, but that of 125-I-corticotropin-(11-24)-tetradecapeptide (125-I-ACTH-1-24 is inhibited by ACTH-1-24 and corticotropin-(1-10)-decapeptide (ACTH-1-10), but ACTH-11-24 at the same molar concentration has no effect. On the other hand, the degradation of 125-I-ACTH-11-24 is protected by ACTH-11-24 and ACTH-1-24, but not by ACTH-1-10. This suggests two systems of degradation, one will have the NH-2-terminal sequence of ACTH-1-24 as substrate, and the other the 11-24 COOH-terminal sequence. The main label product from the degradation of the 125-I-ACTH-1-24 and 125-I-ACTH-11-24 behaves as [125-I]monoiodotyrosine on Sephadex G-50 and paper chromatography. The independence of ACTH binding to its receptor and degradation is demonstrated by the following facts. (a) Calcium and pancreatic trypsin inhibitor completely inhibit the binding at concentrations when the degradation is not altered; (b) the sequences of peptides of ACTH which inhibit the binding and degradation of 125-I-ACTH-1-24 are different.

Recently it has been shown that at the time of the inter-reaction of certain polypeptide hormones with membranes from target tissues, there are two simultaneous but independent processes. One is the binding of the hormone with its receptor; the other is the inactivation of the hormone (l-5).
After purification on a column and in the presence of 0.1 mg of membrane protein, the percentage of binding of r*6I-ACTHll.~r and ~~~I-NPS-ACTH~.Z~ is similar to that of V-ACTH1-2, (60 to 75q7,). On the other hand, if the purification was made by adsorption to Quso G-32 (ll), only 20 to 30yo of the radioactivity was bound to the membranes. In addition, when the specific activities of the three iodinated ACTHs were measured by an antiserum that reacts specifically with the COOH-terminal sequence of ACTH1.9, (The Radiochemical Centre, Amersham, England), the activities were similar (300 to 400 &i per pg).
Binding Measurement of IesZ-ACTH to Membranes-Binding of labeled ACTH to adrenal membranes was performed as previously described (13). Briefly, the membranes were incubated 30 min at 4" in 0.25 ml of 26 mM Tris-HCl buffer (pH 7.4) containing 1% albumin and W-ACTH.
At the end of the incubation the medium was layered over 1 ml of 20 mM Tria-HCl buffer (pH 7.4), 0.25 M sucrose, and 2'$& albumin, and centrifuged immediately in plastic conical tubes at 50,006 X g for 10 min at 0". After aspiration of the supernatant, the tip of the centrifuge tube was cut ofI and counted. All binding determinations were performed in triplicate. Three additional samples were also measured in the presence of unlabeled ACTH (200 pg per sample). The latter estimated the nonspecific binding which was subtracted in each instance. Ten milligrams of ACTH1.14 were labeled with 0.5 mg of Nalz71 and 26 &i of Na1261. The products of the reaction were applied on a carboxymethylcellulose column (1 X 13 cm). The column was eluted as described under "Experimental Procedure." Unlabeled ACTH was measured by its ASW.
cubated in 1 ml of 20 mM Tria-HCI (pH 7.4) containing 1% albumin and labeled ACTH at temperatures and times stated in figures and tables. At the end of incubation the medium was layered over 2.5 ml of 20 mM Tris-HCl (pH 7.4), 0.25 M sucrose, and 2yo albumin, and centrifuged at 50,000 X g for 20 min at 0". Only the 1st ml of the supernatant was aspirated and kept (unbound fraction). The pellet was washed once with 2 ml of the same buffer and centrifuged at 50,000 X g for 20 min. The supernatant was discarded and then the pellet was taken up in 0.5 to 1.0 ml of 1% acetic acid, shaken for 30 min at 22", and centrifuged at 50,000 X g for 10 min. The supernatant (bound fraction) was removed and neutralized with 1 N NaOH.
Between 70 and 30% of the radioactivity present in the pellet was recovered in the acetic acid. For kinetic studies 0.2 ml of the incubation mixture was layered over 0.15 ml of 20 mM Tris-HCl (pH 7.4), 0.25 M sucrose, and 2% albumin contained in a plastic micro-test tube and centrifuged in a Beckman micro-centrifuge.
In these instances, only the degradation of the unbound fraction was studied.
The degradation of the hormone, in the bound and unbound fractions, was studied by the following methods: (a) paper electrophoresis using the system described by Berson and Yalow (15); (b) absorption of the radioactivity on talc (25 mg) in 1 ml of 0.01 M sodium phosphate buffer (pH 7.4) containing 0.2% albumin; (c) absorption of the radioactivity on Quso G-32 (25 mg) in 1 ml of the buffer described above; (d) specific binding to crude adrenal membranes (20,000 X g pellet), at 4' for 30 min as described above. In the last three, measurements were made in triplicate.
In all of the studies, the spontaneous degradation of the hormone was measured in a control sample incubated in the same buffer but without the membrane fraction.
After incubation for 1 hour, there was no degradation at 4", but at 37" the degradation was between 3 and 5'j&. The results are expressed as the percentage of hormone remaining intact compared to the control.
Leucine-aminopeptidase degradation of iodinated ACTH was performed according to the procedure of Lefkowite et al. (9). The nature of labeled products of W-ACTH degradation was studied by gel filtration on a Sephadex G-50 column and by paper chromatography in the system n-butyl alcohol-acetic acid-water (4:1:5) (16).
Adenylate cyclase activity was measured as described elsewhere (14).

RESULTS
Degradation o~'~~I-ACTH~_~~ by Ovine, Rat, and Human Adrenal Preparations-1261-ACTH specifically bound to the crude adrenal membranes of the three species (Table I). Moreover the adenylate cyclase activity of these preparations was stimulated by ACTH (14). These results testify to the presence of ACTH receptors in those adrenal preparations.
After incubating 1251-ACTH 1-24 for 30 min with those fractions the four methods used showed a degradation of unbound lz51-ACTH1.z4 (Table II).
The binding to fresh membranes gave the highest values of degradation.
The degradation at 37" was greater than that observed at 4". In contrast to the unbound ACTH, the bound lz51-ACTH1-2r was not degraded. After dissociation of lz51-ACTH1-24 from its receptors, purity estimated by binding to fresh membranes was higher than the initial 1251-ACTH,-2, (Table II). ACTH is degraded by all of the subcellular fractions of the adrenal (Table III).
However, the most powerful degrading action was in the 20,000 x g pellet (crude membranes). Membranes purified by the method of Finn et al. (11) had a specific activity for degradation, less than that of crude membranes, whereas their specific activity for binding was increased.
These results suggest that the binding and degradation occur at different sites.
Due to the difficulties of obtaining sufficient quantity of highly purified human and rat adrenal membranes, the degradation studies were all done with crude membranes.
Time Course of Degradation of i251-ACTH1-24-The degradation of 1251-ACTH1-24 by adrenal crude membranes is time-and temperature-dependent (Fig. 3). After 10 min of incubation, Concentrations-The degradation of lz51-ACTH1-24 depends on the concentration of membrane proteins (Fig. 4). At concentrations as low as 40 pg per ml, after 30 min of incubation 5 and 30% of the hormone were degraded at 4 and 37", respectively. Under the same conditions, 20 and 9% of the ACTH were specifically bound to the membranes. Given that the binding at 4' is greater than at 37" (13) and that the bound ACTH is protected from degradation, the total quantity of ACTH degraded at 37" is several times greater than at 4".
The amount of ACTHr.Qa degraded (per cent of degraded l*QI-ACTHr-Q4 measured by adsorption to Quso times amount of substrate) is plotted against the substrate concentration.
the binding of lz51-ACTH1.Q4 is inhibited in a similar fashion by ACTHi.
and NPS-ACTHi-24, but the displacement produced by ACTHii-24 and ACTHlalo at the same molarity is respectively about 6 and 15 times less than that produced by ACTH1.2d (Fig. 6). These results suggest that the sequences of ACTH which inhibit the fixation and degradation of 'z51-ACTHi.24 are different. ACTHL10 inhibits the degradation almost as well as ACTHi+ but its power of displacing the binding of 1251-ACTHI.24 is about 15 times less than ACTHim2(. On the other hand, ACTHi,, is much more effective than ACTH1-io for inhibiting the binding of lz51-ACTH1-24 and has a negligible effect on degradation. Efects of ACTH Analogues on Degradation of 1251-ACTH11-24 (Table V) Degradation of ACTH Analogues-A comparative study of the degradation of the three labeled ACTHs (Table IV) shows that at both temperatures, 4 and 37", the degradation of the NPS-ACTHr.24 is similar to that of the ACTHi-24. In contrast the degradation of ACTHll-z4 is much greater.
Effects of ACTH Analogues on Degradation of 1251-ACTH1-24-Degradation of 1251-ACTH~.24 at 4' has been studied in the presence of NPS-ACTHr.24, ACTHi+ ACTHii-24, and ACTHi.io (2.6 x 1W5 M). ACTHI-and NPS-ACTH,L~~ inhibited the degradation of l*rI-ACTH1-24 in a similar fashion, but ACTHii-24 two peptides was greater than that produced by ACTH11-2a was without effect. By contrast, ACTHi-is almost as effec-while ACTHi-10 at high concentrations (5 x 10F4 M) was without tive as the first two analogues (Table V). On the other hand, effect (Fig. 7). Degradation Products of 1251-ACTHl-24 and 1251-ACTH11.24-The degradation products of these two derivatives of ACTH were analyzed by filtration on Sephadex G-50 and paper chromatography. After gel filtration, the main radioactive degradation product is eluted with the same elution volume as [1251]monoiodotyrosine (data not shown). Paper chromatography (Fig. 8) confirmed this result and clearly demonstrated the absence of diiodotyrosine as a degradation product. With some adrenal preparations, a small peak of radioactivity, less polar than diiodotyrosine, which could not be identified represented 10 to 15% of the total radioactivity. Fig. 8 shows that the degradation of 1251-ACTHii-24 measured by the quantity of monoiodotyrosine liberated is greater than that of 1251-ACTHi.24.
Comparative Study of the Degradation of lz51-ACTH1-24 and 1251-ACTH~1.24 by Leucine Aminopeptidase and Adrenal Preparations (Table VI) 8. Crude membranes from human adrenals (1.2 mg per ml) were incubated 40 min with either 1261-ACTH1.2r (left), or ~Z~I-ACTH~I.~~ (right), at 4" (top), or 37" (bottom). After centrifugation a 0.2-ml aliquot of the supernatant was applied to Whatman No. 3MM paper and run in n-butyl alcohol-acetic acid-water system. Labeled ACTHs remain at the origin of the chromatogram. In the same experiments, the percentage of hormone remaining intact, estimated by binding to fresh crude membranes at 4 and 37" was 30 and 3% for iz61-ACTHi.~a, and 11 and 0% for '=I-ACTHii.24.  The results are expressed as per cent of the total radioactivity recovered in the paper.
activity was in the form of monoiodotyrosine. As this compound is the main degradation product of 1251-ACTHi.24 and 1251-ACTHn.24 when incubated with adrenal preparations, we have made a comparative study of these two systems of degradation. Adrenal crude membranes liberate monoiodotyrosine more rapidly from ACTHn-24 than ACTH1.24. The opposite was seen with leucine aminopeptidase. In fact, after 30 min of incubation with leucine aminopeptidase, iz51-ACTHn-2, completely lost its ability to bind to adrenal fresh membranes while only 7 'j$n of monoiodotyrosiue was liberated. This suggests that the enzyme attacks the peptide sequence required for binding, probably lysine 11, before liberating tyrosine 23, Comparative Study of the Binding and Degradation of lt51-ACTH1.zp-The objective was to obtain new arguments which would enable the binding and degradation to be separated. These two processes have been measured using the same adrenal preparation; the results are given in Table VII. Calcium and pancreatic trypsin inhibitor in the concentrations indicated completely inhibited the binding without altering the degradation. Glucagon, however, reduced the degradation but had no effect on the binding.

DISCUSSION
In three species, our results testify to ACTH degradation in subcellular adrenal preparations containing specific ACTH receptors whose existence was proved by 1251-ACTH binding and adenyl cyclase activity.
However, several arguments suggest that ACTH binding and degradation are independent processes: a) the maximum specific activity of the degradation system is in the 20,000 X g pellet and the maximum binding is to purified membranes; b) calcium and pancreatic trypsin inhibitor completely inhibit the binding at concentrations that do not affect the degradation; c) ACTNil. displaces the binding of 1251-ACTH1.2r but has no effect on its degradation, although ACTHL,~ which has only a weak effect on the binding, inhibits the degradation. This type of independence of the phenomena of binding and degradation has been described previously for insulin (2)) glucagon (1,3,5)) and calcitonin (4).
The degradation system of adrenal fraction seems to be different and more complex than leucine aminopeptidase, though monoiodotyrosine is the main radioactive product of both labeled ACTH. The degradation of 1251-ACTHll-za produced by crude membranes (measured by its ability to bind to fresh membranes) is accompanied by a liberation of monoiodotyrosine that is more rapid and greater than the degradation produced by leucine aminopeptidase. Given that tyrosine in position 23 does not take any part in the binding (lo), the degrading system should attack ACTHii-ra either at several points on the molecule or at the NHz-terminal. In the latter case, the degrading system should liberate the amino acids between lysine 11 and tyrosine 23 more rapidly than leucine aminopeptidase.
The kinetic study of the degradation of both ACTHs by leutine aminopeptidase is the hydrolysis of serine 1, once the serine is removed, tyrosine 2 is liberated rapidly (17). Study of the degradation of 1251-ACTH1.~4 and 1251-ACTHn+ shows that for ACTHi-there is a relationship between the diminution of ability to bind and the liberation of labeled monoiodotyrosine; by contrast, for ACTHI~-~( the first process is far more marked than the second. This strongly suggests that the iodotyrosine in these two ACTH analogues is in different positions. In ACTHi1.2a iodotyrosine is obligatory in position 23; in ACTH1.24 the iodination is mainly in position 2. Studies of ACTH structure-function relationship made with several analogues (10,11,(18)(19)(20)(21)(22) have shown that peptide sequence necessary for the binding is located in the COOH-terminal fragment-11-24. Our study agrees with these findings but also suggests that the sequence l-10 NHz-terminal, in addition to being the active biological site of the hormone, could contribute to binding since the affinity of ACTHii-24 (Figs. 6 and 7) for the adrenal receptor is much weaker than that of ACTHLm2d. In addition, ACTHl.lo at high concentrations is able to displace the bound 12SI-ACTH1-24.
Using various ACTH analogues when studying ACTH degradation, we have found data suggesting the existence of at least two enzymatic degradation systems. The degradation of iz51-ACTHi-24 produced by adrenal particulate fractions is protected by ACTHl.24 and ACTHI.,, but not by ACTHn-24. On the other hand, the degradation of 1251-ACTHn.24 is inhibited by ACTHn-24 and ACTHib2+ whereas ACTHlmlo is without effect. The first system should have the NHz-terminal sequence of ACTHI-as substrate; the second the sequence 11-24. This second enzyee system could degrade ACTHi-z(; but either it attacks the peptide molecule after the first system or its affinity for ACTHi-is very low. The physiological significance of the results is unknown. However, we must take ACTH degradation into account to interpret data of ACTH binding and adenylate cyclase stimulation. Moreover ACTH degradation might also explain that isolated adrenal cells' response to ACTH led to cyclic adenosine 3':5'monophosphate and corticosterone productions of which the Hill coefficient is above unity (23) since ACTH is also degraded in rat and sheep isolated adrenal cells preparation2