- Email: [email protected]
Peptide T from human immunodeficiency virus envelope does not interact with hepatic, intestinal and colonic vasoactive intestinal peptide (VIP) receptors
Peptides, Vol. 9, pp. 425-428. ©Pergamon Press plc, 1988. Printed in the U.S.A.
01%-9781/88 $3.00 + .00
BRIEF COMMUNICATION
Peptide T From Human Immunodeficiency Virus Envelope Does Not Interact With Hepatic, Intestinal and Colonic Vasoactive Intestinal Peptide (VIP) Receptors T O A N D. N G U Y E N la
Division o f Gastroenterology, Stanford University School o f Medicbze, Stanford, CA 94305
R e c e i v e d 17 A u g u s t 1987 NGUYEN, T. D. Peptide T from human immunodeficiency virus envelope does not interact with hepatic, intestinal and colonic vasoactive intestinal peptide (V1P) receptors. PEPTIDES 9(2) 425-428, 1988.--Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to specific target cells. An octapeptide sequence contained within the envelope of HIV, peptide T, mediates the viral binding. Since there is a considerable structural homology between peptide T and VIP, it has been proposed that the VIP receptor may be the naturally occurring protein which provides the corresponding cellular attachment site. In three different models (rat intestinal epithelial cell membranes, rat liver plasma membranes and human colonic cells), we document the lack of interaction between peptide T and the VIP receptor. These observations would also exclude any pathophysiologic effect caused by the crossreactivity of peptide T or its analogues and these VIP receptors. AIDS
Human immunodeficiency virus
VIP receptor
H U M A N immunodeficiency virus (HIV) infection is initiated by attachment of the virus to specific target cells. The cellular protein responsible for viral binding has been identified as the CD4/T4 antigen expressed on T4 helper/inducer lymphocytes and accounts for the tissue tropism of HIV [3]. An analogous, if not identical, protein localized to brain membranes seems to mediate HIV infection of the central nervous system [ 11]. On the viral envelope, the peptide sequence responsible for cellular attachment has been identified as an octapeptide sequence called peptide T, contained with the gp 120 envelope glycoprotein [11]. Synthetic peptide T and its analogues, [D-Alal]peptide T amide and pentapeptide T (4--8), inhibit the binding of HIV envelope to rat brain membranes and HIV infection of human T cells [11]; they also trigger human monocyte chemotaxis through the CD4/T4 antigen [12]. Because of an appreciable homology between peptide T and the amino acid sequence 4-11 of vasoactive
Peptide T
intestinal peptide (VIP), it has been postulated by Pert and her colleagues that the CD4/T4 antigen may be the receptor for VIP [12]. If this was the case, the diarrhea observed in the acquired immunodeficiency syndrome (AIDS) may result from the interaction between peptide T from the HIV envelope and the intestinal VIP receptors, triggering cAMP-mediated intestinal secretion [13]. Peptide T and the [D-AlaX]peptide T amide analogue have been tried successfully as therapeutic agents against AIDS [14]. With an initial clinical trial using [D-AlaL]peptide T amide in progress, clarification of the interaction between peptide T and intestinal or hepatic VIP receptors is further warranted. Can peptide T or its analogues cause watery diarrhea by interacting with intestinal or colonic VIP receptors? Will peptide T interact with hepatic VIP receptors, leading to an efficient clearance by the liver in a manner similar to VIP [4]? VIP receptors on rat small intestinal epi-
IRequests for reprints should be addressed to Toan D. Nguyen, M.D., Floor 2, Building 2, V.A. Hospital, 508 Fulton Street, Durham, NC 27705. ZRecipient of NIH Clinical Investigator Award AM 01283.
425
426
NGUYEN
100
% max=mal specific
a
VIP Pept~de T Peptlde T amlde Peptide T (4-8) Secretm
60
binding 40
• 20-
% maximal specific binding
60
® ER-G
O- -II • , -11 -10 -9
,
-8
-6
40
VIP Peptide T
20 ¸ 0-
.
-7
80
0
-5
-11 -10
-9
-8
-7
-6
-5
log unlabelled pepbde
log unlabelled peptide
FIG. 1. Competitive inhibition of VIP binding to rat liver plasma membranes by peptide T, its analogues and secretin. Trace (2.5×10-" M) radioactive 12'~I-VIPwas bound to rat liver plasma membranes in the presence of increasing amounts of unlabeled VIP, peptide T, [D-Alal]peptide T amide, peptide T(4-8) and secretin as outlined in the Method section. For each concentration of unlabelled peptide, the specific binding of radioactive VIP is expressed as a percent of the maximal specific binding. The means from three different experiments are shown (with 10-11 M VIP only one determination was made). For clarity, only the standard errors of the mean (SEM) corresponding to VIP and peptide T are shown. thelial cell membranes [7], rat liver plasma membranes [10] and in the human colonic adenocarcinoma cell line HT 29 [8] have been characterized. This report documents the lack of interaction between peptide T and its analogues with the VIP receptors in these three different models. METHOD
Chemicals, Membrane Preparation and Cell Culture VIP, secretin and bovine serum albumin were obtained from Sigma and peptide T and its analogues (peptide T(4-8), [D-Alal]peptide T amide) from Peninsula Labs. 12~I-VIP, monoiodinated at position 10, was provided by Amersham. Male Sprague Dawley rats (300-350 g) fasted overnight were sacrificed. Liver plasma membranes were prepared according to steps 1-10 of the method outlined by Neville [5]. Intestinal membranes [endoplasmic reticulum (ER)-Golgi and laterobasal membranes (LBM)] were prepared according to a novel method [6] which allowed 9-fold enrichment of ER-Golgi and 15-fold enrichment of LBM. HT 29, a well-differentiated human colonic adenocarcinoma cell line obtained from the American Type Culture Collection was grown in 25 cm 2 flasks at 37°C and 10% CO2. Dulbecco's modified Eagle's minimal essential medium supplemented with 4.5 g/1 glucose, 15% (v/v) fetal bovine serum, 100 U/ml penicillin, 100/zg/ml streptomycin and 0.1 mM non-essential amino acids (all from UCSF Cell Culture Facility, San Francisco, CA) was used to sustain cell growth.
VII) Binding VIP binding to rat intestinal and liver plasma membranes followed procedures detailed previously [10]. 12~I-VIP binding and internalization by HT 29 cells were assessed as described [8]. Confluent colonic cells were exposed to trace radioactive VIP (2.5× 10-" M) in 1 ml of 100 mM sodiumpotassium phosphate buffer (NaH2PO4 and K2HPO4), 1.4% bovine serum albumin, 0.05% Triton X-100, 4.5 g/1 glucose, pH 7.5 (buffer A), for ! hour at 22°C. The medium containing
•
loo "t % maximal specific binding
t
80 60
(~)
40
LBM
20
le~t,de T
0. - i h 0
,
-11 1 0
, -9
-8
• 7
-6
5
log unlabelled peptide
F I G 2 Competitive inhibition of VIP binding to rat intestinal epithelial cell membranes by peptide T. Trace (2.5× 10-'1 M) radioac-
tive 125I-VIPwas bound to rat intestinal epithelial cell membranes in the presence of increasing amounts of unlabeled VIP or peptide T as outlined in the Method section. For each concentration of unlabelled peptide, the specific binding of radioactive VIP is expressed as a percent of the maximal specific binding. The means from three different experiments and the corresponding SEM are shown (with 10 " M VIP only one determination was made). Panel A corresponds to ER-Golgi and panel B to LBM. unbound VIP was then removed and the cells washed sequentially with 1 ml of buffer A, 1 ml of 10 mM sodium phosphate buffer, 0.9% NaCI, pH 7.5 and 1 ml 0.9% NaC1. VIP bound to the cell surface was next removed with exposure to 3 ml of 50 mM glycine-HC1, 100 mM NaCI, pH 2.5 for 5 min and the cells washed with 1 ml of the buffer A. VIP internalized within cells were determined by dissolving the cells in 1 ml 1 M NaOH and washing the flasks with 2 ml 0.9% NaC1. Competitive inhibition of binding was studied by adding different concentrations of either unlabelled VIP, peptide T, peptide T(4-8), [D-Alaqpeptide T amide or secretin to the trace radioactive VIP during the binding incubation. RESULTS
VIP Binding to Liver Plasma Membranes Figure 1 shows the effect of different concentrations of unlabelled VIP, secretin, peptide T and its analogues on the binding of trace radioactive VIP to liver plasma membranes. As previously reported [10], unlabelled VIP competed effectively with a half-maximal inhibition in the nanomolar range.
PEPTIDE T I N T E R A C T I O N W I T H VIP RECEPTOR
427
There was a lesser affinity of the receptor for secretin, with 200 nM required to inhibit VIP binding by 51)%. In contrast, high concentrations (10 -5 M) of either peptide T or the analogues peptide T(4-8) and [D-Alaqpeptide T amide did not affect the binding of radioactive 125I-VIP to liver plasma membranes. This lack of competition with VIP suggests that peptide T and its analogues do not interact with the liver plasma membrane VIP receptor. VIP Binding to Intestinal Epithelial Cell Membranes
In a previous study, we also characterized different VIP receptors on the ER-Golgi and LBM of the rat small intestinal epithelial cell [7]. The receptors located on both membranes demonstrated a high affinity of 1 nM for VIP and a lesser affinity for secretin (10 7 M). Figure 2 shows that in contrast to VIP, peptide T fails to inhibit the binding of the trace radioactive VIP to either ER-Golgi (panel A) or LBM (panel B). There was also no inhibition produced by the analogues of peptide T, [D-Alaqpeptide T amide and peptide T(4--8) (data not shown). VIP Binding and Internalization by H T 29
Using a mild acid wash to differentiate between VIP bound to the surface and internalized within the cell, we have been able to demonstrate that at 22°C, VIP binds to HT 29 and is later internalized within the colonocyte. This internalization was receptor dependent and, along with VIP binding, was specific for VIP with a half-maximal inhibition produced by 1 nM of unlabelled VIP [8]. In contrast, 10-SM of unlabelled peptide T or of its analogues could only inhibit trace VIP binding by approximately 20%. When internalization was considered, there was no inhibition produced by peptide T and its analogues, even at concentrations of up to 10 -5 M (data not shown). DISCUSSION Peptide T has been identified as the portion of the HIV envelope responsible for viral attachment to the cell. Since there is a considerable structural homology between peptide T and VIP (5 of the 8 amino acids are identical), it has been proposed that the VIP receptor may be the corresponding protein naturally occurring on the cell surface which mediates HIV attachment [12]. This postulate however does not take into account the localization of the VIP receptor on tissues that are not specifically infected by HIV (e.g., liver, intestinal mucosa, different smooth muscles and pancreas). To explore directly the validity of this hypothesis, we determined whether peptide T and its analogues could interact with VIP receptors in different tissues by assessing their ability to competitively inhibit VIP binding to these receptors. With rat liver plasma membranes, in contrast to VIP and secretin, neither peptide T nor the analogues [D-
Ala']peptide T amide and peptide T(4--8) could compete against radioactive VIP for membrane binding. The hepatic VIP receptors are responsible for the efficient portal clearance of VIP [4], but considering this lack of interaction it is unlikely that they will mediate the hepatic processing of either the HIV envelope [D-Ala']peptide T amide. Couvineau and Laburthe [1] and Nguyen and Gray [7] have demonstrated structural differences between the VIP receptors in rat intestine and liver plasma membranes. Nguyen and Gray further observed that the intestinal VIP receptor on the LBM is slightly different from the one located on ER-Golgi membranes [7]. It is possible that peptide T may interact with one or more of these different receptors. However, when we evaluated the ability of peptide T and its analogues to inhibit VIP binding to intestinal ER-Golgi or LBM, none of the peptides interacted with the VIP receptors located on these membranes. Considering its marked human tropism, HIV may not recognize the rat VIP receptor. HT 29, a human-derived colonic adenocarcinoma cell line, possesses VIP receptors which can mediate cAMP production and are identical to the receptors found on human colon [2]. Even in this cell line there was little interaction between peptide T and the VIP receptor. The intestinal and colonic VIP receptors will therefore not mediate HIV binding by interacting with peptide T. Furthermore, occupied VIP receptors, coupled with adenylate cyclase, induce electrolyte secretion into the gut lumen, resulting in watery diarrhea. As peptide T does not interact with the intestinal and colonic VIP receptors, this mechanism can neither explain the diarrhea observed occasionally in AIDS nor will it be the basis for an adverse diarrheal side effect produced during therapy with [D-Ala']peptide T amide. In conclusion, in three different models, we observed no demonstrable crossreactivity between peptide T and the rat hepatic and intestinal and human colonic VIP receptors. Without definitively excluding it, these observations do not support the hypothesis that VIP receptors are the naturally occurring proteins that mediate H1V attachment. It is still possible that receptors for the other peptides in the VIP family (secretin, glucagon, GIP, GhRF, thymosin al), may mediate HIV binding, even though they have less similarity with peptide T than VIP. Of note, thymosin a~, which possesses a homology with peptide T and has a role in the immune regulation of T lymphocytes, may be a likely candidate [9]. Finally, we can exclude any pathophysiologic effect produced by peptide T and its analogues resulting from their interaction with the VIP receptors on liver, intestines and colon. ACKNOWLEDGEMENTS The author thanks Dr. Gary M. Gray for his advice and review of the manuscript and Dr. Annabella Iliescas for her diligence in cell culture and membrane preparation.
REFERENCES 1. Couvineau, A. and M. Laburthe. The rat liver vasoactive intestinal peptide binding site. Biochem J 225: 472-479, 1985. 2. Couvineau, A., M. Rousset and M. Laburthe. Molecular identification and structural requirement of vasoactive intestinal peptide receptors in the human colon adenocarcinoma cell line, HT 29. Biochem J 231: 139-143, 1985. 3. Dalgleish, A. G., P. C. L. Beverley, P. R. Clapham, D. H. Crawford, M. F. Greaves and R. A. Weiss. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312: 763-767, 1984.
4. Misbin, R. I., M. M. Wolfe, P. Morris, S. J. Buynitzky and J. E. McGuigan. Uptake of vasoactive intestinal peptide by rat liver. Am J Physiol 243: G103-Glll, 1982. 5. Neville, D. M. Isolation of an organ specific protein antigen from cell surface membrane of rat liver. Biochim Biophys Acta 154: 540-552, 1968. 6. Nguyen, T. D., J.-P. Broyart, K. T. Ngu, A. Iilescas, A. K. Mircheff and G. M. Gray. Laterobasal membranes from intestinal epithelial cells: Isolation free of intracellular membrane contaminants. J Membr Biol 98: 197-205, 1987.
428 7. Nguyen, T. D. and G. M. Gray. Intestinal vasoactive intestinal polypeptide (VIP) receptor: Distinct molecular forms on intracellular membranes and at the enterocyte surface. Clin Res 34: 444A, 1986. 8. Nguyen, T. D., K. T. Ngu and A. Illescas. Vasoactive intestinal peptide: specific binding and internalization by a colonic adenocarcinoma cell line. Gastroenterology 92: 1553, 1987 (abstr.). 9. Nguyen, T. D. and L. A. Scheving. Thymosin al: amino acid homology with peptide T from the human immunodeficiency virus envelope. Biochem Biophys Res Commun 145: 884-887, 1987. 10. Nguyen, T. D., J. A. Williams and G. M. Gray. Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein. Biochemistry 25: 361-368, 1986.
NGUYEN
11. Pert, C. B., J. M. Hill, M. R. Ruff, R. M. Berman, W. G. Robey, L. O. Arthur, F. W. Ruscetti and W. L. Farrar. Octapeptides deduced from the neuropeptide receptor-like pattern of antigen T4 in brain potently inhibit human immunodeficiency virus receptor binding and T-cell infectivity. Proc Natl Acad Sci USA 83: 9254-9258, 1986. 12. Ruff, M. R.. B. M. Martin, E. I. Ginns, W. L. Farrar and C. B. Pert. CD4 receptor binding peptides that block HIV infectivity cause human monocyte chemotaxis. FEBS Left 211: 17-22, 1987. 13. Schwartz, C. J., D. V. Kimberg, H. E. Sheerin, M. Field and S. I. Said. Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J Clin Invest 54: 536--544, 1974. 14. Wetterberg, L., B. Alexius, J. Saaf, A. Sonnerborg, S. Britton and C. Pert. Peptide T in treatment of AIDS. Lancet 1: 159, 1987.
01%-9781/88 $3.00 + .00
BRIEF COMMUNICATION
Peptide T From Human Immunodeficiency Virus Envelope Does Not Interact With Hepatic, Intestinal and Colonic Vasoactive Intestinal Peptide (VIP) Receptors T O A N D. N G U Y E N la
Division o f Gastroenterology, Stanford University School o f Medicbze, Stanford, CA 94305
R e c e i v e d 17 A u g u s t 1987 NGUYEN, T. D. Peptide T from human immunodeficiency virus envelope does not interact with hepatic, intestinal and colonic vasoactive intestinal peptide (V1P) receptors. PEPTIDES 9(2) 425-428, 1988.--Acquired immunodeficiency syndrome (AIDS) is initiated by the attachment of the human immunodeficiency virus (HIV) to specific target cells. An octapeptide sequence contained within the envelope of HIV, peptide T, mediates the viral binding. Since there is a considerable structural homology between peptide T and VIP, it has been proposed that the VIP receptor may be the naturally occurring protein which provides the corresponding cellular attachment site. In three different models (rat intestinal epithelial cell membranes, rat liver plasma membranes and human colonic cells), we document the lack of interaction between peptide T and the VIP receptor. These observations would also exclude any pathophysiologic effect caused by the crossreactivity of peptide T or its analogues and these VIP receptors. AIDS
Human immunodeficiency virus
VIP receptor
H U M A N immunodeficiency virus (HIV) infection is initiated by attachment of the virus to specific target cells. The cellular protein responsible for viral binding has been identified as the CD4/T4 antigen expressed on T4 helper/inducer lymphocytes and accounts for the tissue tropism of HIV [3]. An analogous, if not identical, protein localized to brain membranes seems to mediate HIV infection of the central nervous system [ 11]. On the viral envelope, the peptide sequence responsible for cellular attachment has been identified as an octapeptide sequence called peptide T, contained with the gp 120 envelope glycoprotein [11]. Synthetic peptide T and its analogues, [D-Alal]peptide T amide and pentapeptide T (4--8), inhibit the binding of HIV envelope to rat brain membranes and HIV infection of human T cells [11]; they also trigger human monocyte chemotaxis through the CD4/T4 antigen [12]. Because of an appreciable homology between peptide T and the amino acid sequence 4-11 of vasoactive
Peptide T
intestinal peptide (VIP), it has been postulated by Pert and her colleagues that the CD4/T4 antigen may be the receptor for VIP [12]. If this was the case, the diarrhea observed in the acquired immunodeficiency syndrome (AIDS) may result from the interaction between peptide T from the HIV envelope and the intestinal VIP receptors, triggering cAMP-mediated intestinal secretion [13]. Peptide T and the [D-AlaX]peptide T amide analogue have been tried successfully as therapeutic agents against AIDS [14]. With an initial clinical trial using [D-AlaL]peptide T amide in progress, clarification of the interaction between peptide T and intestinal or hepatic VIP receptors is further warranted. Can peptide T or its analogues cause watery diarrhea by interacting with intestinal or colonic VIP receptors? Will peptide T interact with hepatic VIP receptors, leading to an efficient clearance by the liver in a manner similar to VIP [4]? VIP receptors on rat small intestinal epi-
IRequests for reprints should be addressed to Toan D. Nguyen, M.D., Floor 2, Building 2, V.A. Hospital, 508 Fulton Street, Durham, NC 27705. ZRecipient of NIH Clinical Investigator Award AM 01283.
425
426
NGUYEN
100
% max=mal specific
a
VIP Pept~de T Peptlde T amlde Peptide T (4-8) Secretm
60
binding 40
• 20-
% maximal specific binding
60
® ER-G
O- -II • , -11 -10 -9
,
-8
-6
40
VIP Peptide T
20 ¸ 0-
.
-7
80
0
-5
-11 -10
-9
-8
-7
-6
-5
log unlabelled pepbde
log unlabelled peptide
FIG. 1. Competitive inhibition of VIP binding to rat liver plasma membranes by peptide T, its analogues and secretin. Trace (2.5×10-" M) radioactive 12'~I-VIPwas bound to rat liver plasma membranes in the presence of increasing amounts of unlabeled VIP, peptide T, [D-Alal]peptide T amide, peptide T(4-8) and secretin as outlined in the Method section. For each concentration of unlabelled peptide, the specific binding of radioactive VIP is expressed as a percent of the maximal specific binding. The means from three different experiments are shown (with 10-11 M VIP only one determination was made). For clarity, only the standard errors of the mean (SEM) corresponding to VIP and peptide T are shown. thelial cell membranes [7], rat liver plasma membranes [10] and in the human colonic adenocarcinoma cell line HT 29 [8] have been characterized. This report documents the lack of interaction between peptide T and its analogues with the VIP receptors in these three different models. METHOD
Chemicals, Membrane Preparation and Cell Culture VIP, secretin and bovine serum albumin were obtained from Sigma and peptide T and its analogues (peptide T(4-8), [D-Alal]peptide T amide) from Peninsula Labs. 12~I-VIP, monoiodinated at position 10, was provided by Amersham. Male Sprague Dawley rats (300-350 g) fasted overnight were sacrificed. Liver plasma membranes were prepared according to steps 1-10 of the method outlined by Neville [5]. Intestinal membranes [endoplasmic reticulum (ER)-Golgi and laterobasal membranes (LBM)] were prepared according to a novel method [6] which allowed 9-fold enrichment of ER-Golgi and 15-fold enrichment of LBM. HT 29, a well-differentiated human colonic adenocarcinoma cell line obtained from the American Type Culture Collection was grown in 25 cm 2 flasks at 37°C and 10% CO2. Dulbecco's modified Eagle's minimal essential medium supplemented with 4.5 g/1 glucose, 15% (v/v) fetal bovine serum, 100 U/ml penicillin, 100/zg/ml streptomycin and 0.1 mM non-essential amino acids (all from UCSF Cell Culture Facility, San Francisco, CA) was used to sustain cell growth.
VII) Binding VIP binding to rat intestinal and liver plasma membranes followed procedures detailed previously [10]. 12~I-VIP binding and internalization by HT 29 cells were assessed as described [8]. Confluent colonic cells were exposed to trace radioactive VIP (2.5× 10-" M) in 1 ml of 100 mM sodiumpotassium phosphate buffer (NaH2PO4 and K2HPO4), 1.4% bovine serum albumin, 0.05% Triton X-100, 4.5 g/1 glucose, pH 7.5 (buffer A), for ! hour at 22°C. The medium containing
•
loo "t % maximal specific binding
t
80 60
(~)
40
LBM
20
le~t,de T
0. - i h 0
,
-11 1 0
, -9
-8
• 7
-6
5
log unlabelled peptide
F I G 2 Competitive inhibition of VIP binding to rat intestinal epithelial cell membranes by peptide T. Trace (2.5× 10-'1 M) radioac-
tive 125I-VIPwas bound to rat intestinal epithelial cell membranes in the presence of increasing amounts of unlabeled VIP or peptide T as outlined in the Method section. For each concentration of unlabelled peptide, the specific binding of radioactive VIP is expressed as a percent of the maximal specific binding. The means from three different experiments and the corresponding SEM are shown (with 10 " M VIP only one determination was made). Panel A corresponds to ER-Golgi and panel B to LBM. unbound VIP was then removed and the cells washed sequentially with 1 ml of buffer A, 1 ml of 10 mM sodium phosphate buffer, 0.9% NaCI, pH 7.5 and 1 ml 0.9% NaC1. VIP bound to the cell surface was next removed with exposure to 3 ml of 50 mM glycine-HC1, 100 mM NaCI, pH 2.5 for 5 min and the cells washed with 1 ml of the buffer A. VIP internalized within cells were determined by dissolving the cells in 1 ml 1 M NaOH and washing the flasks with 2 ml 0.9% NaC1. Competitive inhibition of binding was studied by adding different concentrations of either unlabelled VIP, peptide T, peptide T(4-8), [D-Alaqpeptide T amide or secretin to the trace radioactive VIP during the binding incubation. RESULTS
VIP Binding to Liver Plasma Membranes Figure 1 shows the effect of different concentrations of unlabelled VIP, secretin, peptide T and its analogues on the binding of trace radioactive VIP to liver plasma membranes. As previously reported [10], unlabelled VIP competed effectively with a half-maximal inhibition in the nanomolar range.
PEPTIDE T I N T E R A C T I O N W I T H VIP RECEPTOR
427
There was a lesser affinity of the receptor for secretin, with 200 nM required to inhibit VIP binding by 51)%. In contrast, high concentrations (10 -5 M) of either peptide T or the analogues peptide T(4-8) and [D-Alaqpeptide T amide did not affect the binding of radioactive 125I-VIP to liver plasma membranes. This lack of competition with VIP suggests that peptide T and its analogues do not interact with the liver plasma membrane VIP receptor. VIP Binding to Intestinal Epithelial Cell Membranes
In a previous study, we also characterized different VIP receptors on the ER-Golgi and LBM of the rat small intestinal epithelial cell [7]. The receptors located on both membranes demonstrated a high affinity of 1 nM for VIP and a lesser affinity for secretin (10 7 M). Figure 2 shows that in contrast to VIP, peptide T fails to inhibit the binding of the trace radioactive VIP to either ER-Golgi (panel A) or LBM (panel B). There was also no inhibition produced by the analogues of peptide T, [D-Alaqpeptide T amide and peptide T(4--8) (data not shown). VIP Binding and Internalization by H T 29
Using a mild acid wash to differentiate between VIP bound to the surface and internalized within the cell, we have been able to demonstrate that at 22°C, VIP binds to HT 29 and is later internalized within the colonocyte. This internalization was receptor dependent and, along with VIP binding, was specific for VIP with a half-maximal inhibition produced by 1 nM of unlabelled VIP [8]. In contrast, 10-SM of unlabelled peptide T or of its analogues could only inhibit trace VIP binding by approximately 20%. When internalization was considered, there was no inhibition produced by peptide T and its analogues, even at concentrations of up to 10 -5 M (data not shown). DISCUSSION Peptide T has been identified as the portion of the HIV envelope responsible for viral attachment to the cell. Since there is a considerable structural homology between peptide T and VIP (5 of the 8 amino acids are identical), it has been proposed that the VIP receptor may be the corresponding protein naturally occurring on the cell surface which mediates HIV attachment [12]. This postulate however does not take into account the localization of the VIP receptor on tissues that are not specifically infected by HIV (e.g., liver, intestinal mucosa, different smooth muscles and pancreas). To explore directly the validity of this hypothesis, we determined whether peptide T and its analogues could interact with VIP receptors in different tissues by assessing their ability to competitively inhibit VIP binding to these receptors. With rat liver plasma membranes, in contrast to VIP and secretin, neither peptide T nor the analogues [D-
Ala']peptide T amide and peptide T(4--8) could compete against radioactive VIP for membrane binding. The hepatic VIP receptors are responsible for the efficient portal clearance of VIP [4], but considering this lack of interaction it is unlikely that they will mediate the hepatic processing of either the HIV envelope [D-Ala']peptide T amide. Couvineau and Laburthe [1] and Nguyen and Gray [7] have demonstrated structural differences between the VIP receptors in rat intestine and liver plasma membranes. Nguyen and Gray further observed that the intestinal VIP receptor on the LBM is slightly different from the one located on ER-Golgi membranes [7]. It is possible that peptide T may interact with one or more of these different receptors. However, when we evaluated the ability of peptide T and its analogues to inhibit VIP binding to intestinal ER-Golgi or LBM, none of the peptides interacted with the VIP receptors located on these membranes. Considering its marked human tropism, HIV may not recognize the rat VIP receptor. HT 29, a human-derived colonic adenocarcinoma cell line, possesses VIP receptors which can mediate cAMP production and are identical to the receptors found on human colon [2]. Even in this cell line there was little interaction between peptide T and the VIP receptor. The intestinal and colonic VIP receptors will therefore not mediate HIV binding by interacting with peptide T. Furthermore, occupied VIP receptors, coupled with adenylate cyclase, induce electrolyte secretion into the gut lumen, resulting in watery diarrhea. As peptide T does not interact with the intestinal and colonic VIP receptors, this mechanism can neither explain the diarrhea observed occasionally in AIDS nor will it be the basis for an adverse diarrheal side effect produced during therapy with [D-Ala']peptide T amide. In conclusion, in three different models, we observed no demonstrable crossreactivity between peptide T and the rat hepatic and intestinal and human colonic VIP receptors. Without definitively excluding it, these observations do not support the hypothesis that VIP receptors are the naturally occurring proteins that mediate H1V attachment. It is still possible that receptors for the other peptides in the VIP family (secretin, glucagon, GIP, GhRF, thymosin al), may mediate HIV binding, even though they have less similarity with peptide T than VIP. Of note, thymosin a~, which possesses a homology with peptide T and has a role in the immune regulation of T lymphocytes, may be a likely candidate [9]. Finally, we can exclude any pathophysiologic effect produced by peptide T and its analogues resulting from their interaction with the VIP receptors on liver, intestines and colon. ACKNOWLEDGEMENTS The author thanks Dr. Gary M. Gray for his advice and review of the manuscript and Dr. Annabella Iliescas for her diligence in cell culture and membrane preparation.
REFERENCES 1. Couvineau, A. and M. Laburthe. The rat liver vasoactive intestinal peptide binding site. Biochem J 225: 472-479, 1985. 2. Couvineau, A., M. Rousset and M. Laburthe. Molecular identification and structural requirement of vasoactive intestinal peptide receptors in the human colon adenocarcinoma cell line, HT 29. Biochem J 231: 139-143, 1985. 3. Dalgleish, A. G., P. C. L. Beverley, P. R. Clapham, D. H. Crawford, M. F. Greaves and R. A. Weiss. The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature 312: 763-767, 1984.
4. Misbin, R. I., M. M. Wolfe, P. Morris, S. J. Buynitzky and J. E. McGuigan. Uptake of vasoactive intestinal peptide by rat liver. Am J Physiol 243: G103-Glll, 1982. 5. Neville, D. M. Isolation of an organ specific protein antigen from cell surface membrane of rat liver. Biochim Biophys Acta 154: 540-552, 1968. 6. Nguyen, T. D., J.-P. Broyart, K. T. Ngu, A. Iilescas, A. K. Mircheff and G. M. Gray. Laterobasal membranes from intestinal epithelial cells: Isolation free of intracellular membrane contaminants. J Membr Biol 98: 197-205, 1987.
428 7. Nguyen, T. D. and G. M. Gray. Intestinal vasoactive intestinal polypeptide (VIP) receptor: Distinct molecular forms on intracellular membranes and at the enterocyte surface. Clin Res 34: 444A, 1986. 8. Nguyen, T. D., K. T. Ngu and A. Illescas. Vasoactive intestinal peptide: specific binding and internalization by a colonic adenocarcinoma cell line. Gastroenterology 92: 1553, 1987 (abstr.). 9. Nguyen, T. D. and L. A. Scheving. Thymosin al: amino acid homology with peptide T from the human immunodeficiency virus envelope. Biochem Biophys Res Commun 145: 884-887, 1987. 10. Nguyen, T. D., J. A. Williams and G. M. Gray. Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein. Biochemistry 25: 361-368, 1986.
NGUYEN
11. Pert, C. B., J. M. Hill, M. R. Ruff, R. M. Berman, W. G. Robey, L. O. Arthur, F. W. Ruscetti and W. L. Farrar. Octapeptides deduced from the neuropeptide receptor-like pattern of antigen T4 in brain potently inhibit human immunodeficiency virus receptor binding and T-cell infectivity. Proc Natl Acad Sci USA 83: 9254-9258, 1986. 12. Ruff, M. R.. B. M. Martin, E. I. Ginns, W. L. Farrar and C. B. Pert. CD4 receptor binding peptides that block HIV infectivity cause human monocyte chemotaxis. FEBS Left 211: 17-22, 1987. 13. Schwartz, C. J., D. V. Kimberg, H. E. Sheerin, M. Field and S. I. Said. Vasoactive intestinal peptide stimulation of adenylate cyclase and active electrolyte secretion in intestinal mucosa. J Clin Invest 54: 536--544, 1974. 14. Wetterberg, L., B. Alexius, J. Saaf, A. Sonnerborg, S. Britton and C. Pert. Peptide T in treatment of AIDS. Lancet 1: 159, 1987.