Thymic peptide thymosin cq (10 -9 to 3 ⢠10 -7 M) is shown to inhibit the specific binding of [12sI]VIP to rat blood mononuclear cells and liver plasma membranes.
Bioscience Reports, Vol. 6, No. 8, 1986
Interaction of Thymic Peptide Thymosin with Vasoactive Intestinal Peptide (VIP) Receptors J. R. Calvo 1, R. Goberna and J. M. Guerrero Received August 4, 1986
KEY WORDS: Thymosin;vasoactivcintestinalpeptide. Abbreviations:GRF, growth hormone releasingfactor; PHI, porcineintestinalpeptide havingN-terminal histidine and C-terminalisoleucineamide; GIP, gastric inhibitorypolypeptide;VIP, vasoactiveintestinal peptide. Thymic peptide thymosin cq (10 - 9 t o 3 • 10 - 7 M) is shown to inhibit the specific binding of [12sI]VIP to rat blood mononuclear cells and liver plasma membranes. Thymosin 71 was 160 and 6250 times less potent that VIP at inhibiting [~25I]VIP binding to blood mononuclear cells and liver plasma membranes, respectively. Thymosin ~ (10- lo to 10- 7 M) was weak in stimulating adenylate cyclase activity. Its efficacy is about 25 % and 27 % that of native VIP in blood mononuclear cells and liver plasma membranes, respectively. Thymosin ~ may behave as a partial VIP agonist in rat.
INTRODUCTION Thymosin cq was the first polypeptide isolated from thymosin fraction 5, a polypeptide extract of calf thymus (1, 2). This peptide consists of 28 amino acid residues and has a molecular weight (MW) of 3108 (1, 3, 4). The analysis of the sequence of this peptide reveals close structural similarities with the members of the socalled glucagon-VIP-secretin family (5,6). Thymosin el possesses 6 amino acids in common with the rat growth hormone releasing factor (rGRF) (7), 5 with the vasoactive intestinal peptide (VIP) (8), glucagon (9) and porcine gut peptide PHI (6)
Dept. de Bioqulmica,Facultad de Medicina,Avda. SfinchezPizjufin,4, 41009-SEVILLA,Spain. 1 "17owhom correspondenceshould be addressed. 727 0144-8463/86/0800-0727505.00/0 9 1986 Plenum Publishing Corporation
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Calvo, Goberna,and Guerrero
and 3 with secretin (10), gastric inhibitory polypeptide (GIP) (11) and human growth hormone releasing factor (hGRF) (5). Peptides of this structural family exhibit great overlapping biological activities in various tissues (12). This was shown to be due in some cases to cross-reactivity at the receptor level (13-16). The structural similarities between thymosin e~ and VIP prompted us to investigate the possible interaction of thymosin e~ with VIP receptors. In this paper we show that thymosin e~ binds to VIP receptors in rat blood mononuclear cells and liver plasma membranes and stimulates adenylate cyclase activity.
MATERIALS AND METHODS
Materials Synthetic bovine thymosin a~ was purchased from Sigma (St Louis, MO, USA); synthetic VIP was purchased from Peninsula Laboratories (San Carlos, CA, USA); thymosin fraction 5 was generously supplied by Professor M. Freire (Santiago de Compostela, Spain); carrier-free Na [1251] (IMS-30) and 2,8-[all]adenosine 3',5' cyclic phosphate (TRK-498) were obtained from the Radiochemical Centre (Amersham, UK). [12si]Vi P was prepared by the chloramine T method at a specific activity of 250 Ci/g (17) and had binding properties identical to those of native VIP (18). All other chemicals were reagent grade.
Experimental Procedures Peripheral blood mononuclear cells and liver plasma membranes were obtained from Wistar rats as described in (19) and (20), respectively. Studies of [125I]VIP binding to rat blood mononuclear cells and liver plasma membranes were conducted as in (21) and (22), respectively. All values for binding of [I:sI]VIP to rat blood mononuclear cells and liver plasma membranes are expressed as "specific binding", that is total binding minus nonspecific binding. So-called "nonspecific binding" was that binding which occurred in the presence of 10 #M native VIP. These values were always less than 12 % and 8 % for blood mononuclear cells and liver plasma membranes, respectively. Adenylate cyclase activity was assayed exactly as in (21, 23).
RESULTS Thymosin "1 ( 10-9 to 3 x 10- 7 M) inhibits the specific binding of [125I]VIP to its receptors present in rat blood mononuclear cells (Fig. 1, top). Half-maximal inhibition is obtained at 160nM. Thymosin ~1 is 160 times less potent than VIP at inhibiting [12SI]VIP binding to blood mononuclear cells. In rat liver plasma membranes, thymosin a 1 (10 -9 to 3 x 10 -~ M) was less effective in inhibiting the specific binding of [ ~25i]ViP (Fig. 1, bottom). Half-maximal inhibition is obtained at 3500nM and thymosin ~ was 6250 times less potent than VIP at inhibiting [125I]VIP binding to liver plasma membranes.
Thymosin-VIP Receptor Interaction 1
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The effect of thymosin ~tl (10 -1~ to 10 -T M) on cyclic AMP production in rat blood mononuclear cells and liver plasma membranes was tested. In both cases, thymosin ~tl was weak in stimulating adenylate cyclase activity (Fig. 2, top and bottom). These results indicate that thymosin ~1 appears to be a partial VIP agonist
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Fig. 2. Adenylate cyclase activity in rat blood mononuclear cells (top) and liver plasma membranes (bottom) in response to increasing concentrations of VIP (0) and thymosin ~1 (O). Each point is the mean of triplicate determinations. Two other experiments gave similar results.
with low intrinsic activity in blood mononuclear cells and liver plasma membranes since, (1) it is less potent in stimulating adenylate cyclase activity (Fig. 2, top and bottom) than in inhibiting the binding of E125I]VIP (Fig. 1, top and bottom), and, (2)
Thymosin-VIP Receptor Interaction
731
Table 1. Cyclic AMP production in rat blood mononuclear cells Addition
cAMP (pmol/106 cells)
0 VIP (10 -7 M) Thymosin ~1 (10-7 M) VIP (10 - 7 M) + Thymosin cq (10-7 M)
4+1 15 __+3 6+ 2 10 + 2
Results are the mean __+SEM of 4 experiments.
Table 2. Cyclic AMP production in rat liver plasma membranes
Addition
cAMP (pmol/mg protein)
0 VIP (10-8 M) Thymosin ~x (10-7 M) VIP (10-8 M) + Thymosin ~a (10-7 M)
20-t-5 72 + 12 26 ___7 37 + 8
Results are the mean + SEM of 4 experiments.
its efficacy is a b o u t 25 % a n d 27 % t h a t of native V I P in b l o o d m o n o n u c l e a r cells a n d liver p l a s m a m e m b r a n e s , respectively. T h y m o s i n cq a n d V I P , tested at m a x i m a l l y active doses, do n o t elicit an a d d i t i v e s t i m u l a t o r y effect on cyclase activity in rat b l o o d m o n o n u c l e a r cells a n d liver p l a s m a m e m b r a n e s (Tables 1 a n d 2). Table 3. Effectsof native VIP, thymosin ea and thymosin fraction 5 on the specific binding of [12sI]VIP to rat liver plasma membranes
Addition
Specific [I/sI]VIP binding
0 VIP (0.234/~g/ml) Thymosin cq (0.622pg/ml) Thymosin fraction 5 (100#g/ml)
16.7 ___2.4 4.8 + 1.7 15.6 + 3.6 13.0 __+3.1
Results are the mean ___SEM of 4 experiments. In T a b l e 3 we show the effect of t h y m o s i n fraction 5 (100 #g/ml) on the specific b i n d i n g of [125I]VIP to liver p l a s m a m e m b r a n e s . T h y m o s i n fraction 5, at the c o n c e n t r a t i o n tested, inhibits the specific b i n d i n g of [ t 2 5 I ] V I P to its receptors.
DISCUSSION
The present p a p e r clearly d e m o n s t r a t e s t h a t a new peptide, t h y m o s i n ~1, is able to b i n d to V I P r e c e p t o r s in r a t b l o o d m o n o n u c l e a r cells a n d liver p l a s m a m e m b r a n e s .
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Thymosin ~1 is an acidic peptide with a molecular weight of 3108 and an isoelectric point of 4.2, isolated from thymosin fraction 5, a polypeptide extract of calf thymus. Thymosin ~1 possesses 28 amino acid residues and has an N-terminal acetylated group. High concentrations of thymosin ~1 are needed to interact with VIP receptors in rat blood mononuclear cells and liver plasma membranes. In fact, 200 and 6666 times higher concentrations of thymosin ~x were necessary to observe the same effect as with VIP in blood mononuclear cells and liver plasma membranes, respectively. This apparent disagreement between the results obtained with blood mononuclear cells and liver plasma membranes can be explained by the recent evidence of the molecular heterogeneity of VIP receptors (24, 25, 26). Furthermore, thymosin ~i was weak in cyclic AMP production in both cases. It must be kept in mind that the thymosin ~1 used was of bovine origin, while the blood mononuclear cells and liver plasma membranes were of rat origin. In fact, a great species specificity exists in the ability of VIP receptors to discriminate between the different peptides of its structural family (14, 16, 27, 28). In that respect only studies performed in homologous systems are relevant for analysing the structure-function relationship of peptides of the VIP-secretin family. Previous work has shown the importance of the N-terminal portion of VIP in the binding to receptors (18, 30). Thymosin el has 3 amino acids in common with VIP in the 10 first amino acids at the N-terminal (Ala-4, Val-5 and Thr-7) while 2 other amino acids are in common in the remainder of the molecule (Lys-20 and Ash-28). In particular, the threonine located in position 7 has been shown to be very important for binding receptors (18). Although the N-terminal portion is certainly involved in the recognition of thymosin e~ by VIP receptors, the resemblances in the remainder of the peptide sequence cannot be neglected. Indeed, the entire length of the VIP sequence is necessary to the process of binding, since VIP fragments have very low affinity, if any, for the receptors (30, 31). The presence of a N-terminal serine in thymosin el, instead of a histidine as in VIP, is very probably a cause of the low affinity of thymosin cq for VIP receptors. These results are in good agreement with previous observations indicating that deleting (14, 30) or modifying (16, 18, 32, 33) the N-terminal histidine in some of the peptides of the structural family of VIP results in a drastic loss of affinity for rat VIP receptors and generate partial VIP agonists. Furthermore, thymosin st has an acetylated serine Nterminal and it has been shown that a free N-terminal amino group is essential for generating the biological response. Indeed, Ac-Tyr~hGRF is a competitive VIP antagonist in rat and a partial VIP agonist in humans (16). Thymosin fraction 5 is a polypeptide extract of calf thymus. It contains at least 30 different polypeptides, which can be resolved by two dimensional gel electrophoresis or isoelectric focussing. Thymosin el is the first thymosin polypeptide isolated from bovine fraction 5. Several other thymosin peptides have been isolated from fraction 5, for example, thymosin e7 (MW=2000) (4), thymosin /~3 (MW=5500) (34) and thymosin 84 (MW=4982) (34). Our results are in good agreement with previous reports indicating that the yield of thymosin :(1 from fraction 5 is about 0.6 ~ (35). In conclusion, this paper indicates that thymosin el binds to VIP receptors present in rat blood mononuclear cells and liver plasma membranes. Thymosin e~ was weak in stimulating adenylate cyclase activity in both cases and may behave as a partial VIP agonist in rat.
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Thymosin-VIP Receptor Interaction
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