JOURNAL OF MOLECULAR RECOGNITION, VOL. 4.53-56 (1991)
Proposed Loci of Interaction between Bombesin and its Receptor G. L. Tritsch Department of Experimental Biology, Roswell Park Cancer Institute, New York State Department of Health, Buffalo. 14263. USA
The non-coding strand of the bombesin receptor gene, when ‘translated’ 5’ to 3’, contains an interrupted sequence of the 10 amino acids of bombesin. This finding forms the basis for proposing the points of contact between bombesin and its receptor as well as a partial conformation of the binding region of the receptor.
INTRODUCTION
Recognition in the biological context is thought to involve complementary properties at the interacting surfaces. A remarkable hypothesis has been suggested by Blalock and Smith (1984) based on complementary hydropathy of amino acids of interacting polypeptides. The observation that codons for hydrophobic amino acids of a gene are complemented by codons for hydrophylic amino acids in the non-coding DNA strand, and the converse, is the basis of the hypothesis. The intruiging aspect of this idea is that the genetic code has a role in determining the complementarity of interacting polypeptides. It has further been demonstrated (Tritsch, 1989) that comparable hydropathic profiles of polypeptides suggest a common binding activity. The relevance of this to ligand-receptor interactions was demonstrated by Bost et al. (1985a) by showing that regions of complementarity can be predicted from the gene sequence of epidermal growth factor, interleukin-2, and transferrin with their respective receptors. I n their paper, the non-coding strand of the gene was ‘translated’ 3’ to 5‘ (herein designated nc3’-N with the first amino acid residue as the amino terminus), and in this sequence, several penta- and hexapeptide sequences were found which matched sequences in the respective ligand, albeit not with 100% homology: thus, nucleotide homologies between 53% and 78%, and amino acid homologies of 80% and 83% (one wrong residue in a penta- and hexapeptide sequence, respectively) resulted. Because of redundancy within the genetic code, not all inconsistencies in the gene sequence resulted in changes in the amino acid residues. Other examples of this approach can be found in the literature with constructs prepared nc3’-N (ACTH, Blalock and Boot, 1986), nc3’-C (angiotensin, Soffer et af., 1987), ncS’-N (ACTH, Bost et al., 198%; yendorphin, Carr et ul., 1986; RNAase, Shai etal., 1987; angiotensin, Elton et al., 1988; fibronectin, Brentani et al., 1988; insulin, Knutson, 1988; gonadotrophin releasing hormone, Gorcs et ul., 1986) and nc5‘-C (RNAase. Shai et al., 1987; luteinizing hormone releasing hormone, Mulchahey et al., 1986). Since the gene for the receptor of mammalian bombesin-like peptides has been sequenced recently OY52-.34Y9/Y 1/020053-04 $05 .(HI Wiley & Sons, Ltd
0 19Y1 by John
(Battey ef al., 1991) we derived the non-coding strand from it and examined it for homologous amino acid sequences in bombesin.
RESULTS AND DISCUSSION The bombesin receptor, which is a member of the guanine nucleotide binding protein-coupled receptor superfamily, has an extracellular amino-terminal sequence of 39 amino acids, 7 transmembrane domains, and 1 carboxy-terminal intracellular domain. The entire receptor gene sequence was used to derive the sequence of the non-coding strand, and the latter ‘translated’ from the 5‘ and 3’ ends to obtain the nc5’ and nc3‘ constructs, respectively, as outlined by us previously (Markus et al., 1989). Both of these constructs were examined N to C and C to N to determine if bombesin amino acid sequences could be found. No sequences could be found in the nc3‘ constructs, but the nc5’ construct contained the homologies shown in Fig. 1 in t h e amino-terminal extracellular domain where a total of seven correctly spaced homologies occured with one 5 amino acid space, one 2 amino acid space, and two 1 amino acid spaces. The extracellular domain between membrane-spanning domains 4 and 5 contained three amino acids with a 1 amino acid space that matched three correctly spaced amino acids in bombesin not accounted for by the amino-terminal extracellular domain. Thus, of the 14 amino acids in bombesin, 10 amino acids were found in the receptor by ‘translation’ of the non-coding strand of the gene from the 5‘ end. No homologies could be found in the intracellular or transmembrane portions of the receptor. Because it is highly unlikely that such a long segment be linear in the three-dimensional structure of the bombesinbombesin-receptor complex, the three spaces and the four amino acid residues in bombesin for which no corresponding amino acid residues were found in the construct should not be surprising. Since previous findings of homologies with hexapeptides with one mismatched amino acid were shown to be very highly significant statistically (Bost el af., 198%). finding 10 juxtaposable amino acids constitutes convincing evidence that the sequence of these amino acid residues is highly unlikely to be due to chance. Received 29 March 1991 Accepted (revised) 15 July I991
G . L. TRITSCH
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3omoes:n Receptor Gene (Nucieotides 573-586) 3omaes:n Receptor (Extracellular a m a i n k r w e e n memcrzne-soinning comzins C and f :
Figure 1. Genesis of the nc5' construct of the non-coding strand of the bombesin receptor gene. The lines connecting the nucleotides in a given triplet denote canonical Watson-Crick G.C and A.T pairing between the gene and the non-coding strand. The non-coding strand shown was 'translated' 5' to 3' t o produce the amino acids in the nc5' construct. The heavy lines indicate amino acids in the nc5' construct of the gene which are identical to amino acids in bombesin. Note that valine in position 10 of bombesin could be aligned with either of the t w o adjacent valines in the nc5' construct.
If the hydropathic profile of this sequence of 10 amino acids is examined (Fig. 2), it becomes apparent that not all of the 10 juxtaposable amino acids are likely to contribute to binding: interaction between E of bombesin and N of the receptor [both with Kyte and Doolittle (1982) hydropathy scores of - 3.51, between A of bombesin and C of the receptor (hydropathy scores of 1.8 and 2.5, respectively), and between both instances of G of bombesin and T of the receptor (hydropathy scores of - 0.4 and - 0.7, respectively). The hydropathies of these four pairs of amino acids are similar and of the same sign and thus cannot contribute to hydropathic complementarity. When the 10 proposed contact points are used to prepare paired hydropathy plots (Markus et al., 1989) of bombesin and the corresponding amino acids in the receptor (Fig. 3), the results indicate significant hydro-
pathic complementarity ( r = 0.65; slope = 0.67). This level of Complementarity is put into proper perspective when one considers other receptor-ligand interactions. For example, paired hydropathy plots of ACTH and its nc3' and nc5' constructs gave correlation coefficients of 0.79 and 0.61, respectively (Markus et al., 1989). Both of these constructs competed with ACTH in binding experiments in several biological systems with affinity constants in the nanomolar range (Blalock and Bost, 1986). Binding of the nc3' and ncS' constructs of substance P were equivalent (Pascual et al., 1988) as were the correlation coefficients of paired hydropathy plots with substance P ( r = 0 . 8 3 for nc3'; r=0.76 for nc5'). Another example, binding of the nonapeptide vasopressin to its receptor was inhibited only by the nc3' construct; the ncS' construct was inactive (Johnson and Torres, 1988). When the appropriate paired hydro-
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Figure 2. Hydropathic profile of bombesin (H) and its The Kyte and Doolittle (1982) hydropathy scale receptor (a-0).
is used.
Figure 3. Paired hydropathy plot for the 10 amino acids i n bombesin and the amino acids to which they are bound in the receptor (see Fig. 1). The hydropathy scale of Kyte and Doolittle (1982) is used, and the value for an amino acid in the receptor is plotted on the abscissa and the value of the corresponding amino acid i n bombesin is plotted on the ordinate. Negative values to the left and below the origin denote hydrophilic amino acids and positive values, to the right and above the origin, hydrophobic amino acids. The concentric circles indicate that t w o amino acid pairs have identical coordinates. The slope of the least square linear regression shown is -0.67 with a correlation coefficient of 0.65.
PROPOSED INTERACTION LOCI OF BOMBESIN AND ITS RECEPTOR
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Figure 4. Scheme of the bombesin binding domain within the bombesin receptor. (a) The cell membrane is stippled with the extracellular portion on top and the intracellular portion on the bottom. The length of the lines are in proportion to the number of amino acid residues (scale indicated) in each extracellular and intracellular (horizontal lines) domain and transmembrane (vertical lines) domain as deduced from the amino acid sequence (Battey et a/., 1991). In this linear projection, the domains are arranged so that the bombesin binding regions proposed in Fig. 1 can be aligned opposite each other, N-C, to accomodate bombesin, but the lengths of the straight lines which are in direct proportion to the number of amino acids they contain does not allow alignment of the t w o highlighted receptor contact regions as in Fig. 1; the pair of broken lines denotes this displacement. This is obviated in (b). For clarity, bombesin has not been added but is intended t o fit between the t w o highlighted regions in the t w o extracellular domains of the receptor. The transmembrane domains are numbered as i n Battey e t a / . (1991), and 'N' and 'C' denote the amino and carboxy termini of the receptor. It is of interest that the total number of amino acids connecting transmembrane domains 2, 3 and 4 are equal to those connecting transmembrane domains 4 and 5. (b) A view from outside the cell, looking down on the receptor. The extracellular domains are drawn as solid lines and the intracellular domains as broken lines. The same scale as in (a) is used t o denote the number of amino acid residues i n the domains linking the transmembrane domains. Here, the transmembrane domains are arranged in a circle and this allows alignment of the contact points between bombesin and its receptor as shown in Fig. 1. The configuration of the domains between transmembrane domain 5 and the carboxy terminus is arbitrary and unrelated t o the data in Fig. 1.
pathy plots are prepared (data not shown), correlation coefficients of 0.65 and 0.26 for the nc3' and nc5' constructs, respectively, are found. Thus, it becomes apparent that only those constructs which yield amino acid sequences which have correlation coefficients in paired hydropathy plots of ca 0.6 and above should be expected to be active biologically. If the data from the four pairs of amino acids of similar hydropathy of Fig. 2 are removed from consideration (data points in the upper right quadrant and lower left quandrant of Fig. 3 ) . the least square linear regression based on the six remaining juxtaposable amino acid pairs has a slope of - 1 .O1 with a correlation coefficient of 0.997. From this present analysis one would predict that bombesin makes contact with its receptor in the extracellular amino terminal domain as well as with the extracellular domain between the fourth and fifth transmembrane domains (Fig. 4). Here we have complementarity of 10 amino acids. Since the sequence of the bombesin gene is not yet known. we do not know the degree of nucleotide homology which is generally less than amino acid homology due to redundancy within the genetic code. The probability that these amino acid homologies could accur accidentally is unlikely: Dayhoff er a f . (1983) have shown that ' . . . a search for a specific penta- or hexapeptide will usually turn up the source protein and its close relatives or nothing at all'. 'For example, the sequence GDSGG surrounding the serine active site of trypsin is absolutely conserved in all 19 of the sequenced serine proteases related to trypsin, including four bacterial sequences, and it occurs
nowhere else in our present protein sequence data base.' Furthermore, Dayhoff and Orcutt (1979) have stated that 'If a protein sequence is known, a segment can usually be identified with confidence through comparison with the data file of all known sequences when the identity and position of only 7 amino acid residues (not necessarily contiguous) are known'. These findings were confirmed by Bost et al. (1985a) in their analysis of ligand-receptor interactions. The NBRF Protein Information Resource data base ( S O 908 sequence entries) was searched for homologies of the sequence of the bombesin receptor and its nc5' construct which encompasses our proposed loci of interaction (Fig. 1) in both directions, i.e., NLDVDPFLSCNDTFNQ and QNFTDNCSLFPDVDLN. The highest degree of identity found was for 6 out of 16 amino acids (37.5%) as compared to our 10 out of 16 amino acids (62.5%). These lesser homologies were found in rat S-acyl fatty acid synthase thioesterase and rat oleoyl-[acyl-carrierprotein] hydrolase (.LD.. .FL.. .DT...), Escherichia coli NADP-specific glutamate dehydrogenase (NL ...,FL ....TF..) and in mouse and human dihydrofo.FP..DL.). Of all of these, only late reductase (Q.F the dihydrofolate reductase homologies are found when the amino acid sequence was the reverse of that shown in Fig. 1 , i.e., QNFTDNCSLFPDVDLN. The pattern of these homologies differ among themselves as well as from the ones shown in Fig. 1. None of the proteins identified in the search function as receptors, and the positions of homologies are separated by multiple gaps of three or more amino acid residues. Because
Figure 2. Three-dimensional structure of the Ha-ras oncogene product p21 shown as a stereo pair in thea-carbon backbone presentation. The GTP molecule is given in yellow within the active site. The 'hot spots'are identified by the amino acid residuesand Van der Waalssurfaces given in red. All 'hot spots'are clustered in and around the GTP binding active site.
56
G . L. TRITSCH
of this, we did not search the data base for the missing amino acid homologies in other portions of the amino acid sequence of the respective proteins.
amino acids suggests that these regions are involved in the binding of bombesin to its receptor. Crystallographic data, and possibly active site directed mutagenesis studies will confirm or refute this suggestion.
CONCLUSION It is thus concluded that the likelihood that our amino acid homologies could occur accidentally appears to be exceedingly small. This communication is the first demonstration of the use of information derived from the non-coding strand Of a gene Of a sing1e sequence in a ligand binding to non-continuous sequences of a receptor. Thus, finding 10 closely spaced homologous
Acknowledgements The NBRF Protein Information Resource data base search was expertly performed by G . E. Franke, MLS, of the Medical and Scientific Library of this Institute. Appreciation is expressed to Dr Gabor Markus for his valuable discussions during the course of this work and for critical evaluation of this manuscript.
REFERENCES
Battey, J. F.. Way, J. M., Corjay, M. H., Shapira, H.. Kusano, K., Harkins, R., Wu, J. M., Slattery, T., Mann, E. and Feldman, R. I. (1991). Molecular cloning of the bombesinlgastrinreleasing peptide receptor from Swiss 3T3 cells. Proc. Natl. Acad. Sci. USA 88, 395-399. Blalock, J. E. and Smith, E. M. (1984). Hydropathic anticomplementarity of amino acids based on the genetic code. Biochern. Biophys. Res. Cornrnun. 121, 203-207. Blalock, J. E. and Bost, K. L. (1986). Binding of peptides that are specified by complementary RNAs. Biochern. J. 234, 679683. Bost, K. L., Smith, E. M. and Blalock, J. E. (1985a). Regions of complementarity between the messenger RNAs for epidermal growth factor, transferrin, interleukin-2 and their respective receptors. Biochern. Biophys. Res. Cornrnun. 128, 1373- 1380. Bost, K. L., Smith, E. M. and Blalock, J. E. (1985b). Similarity between the corticotropin (ACTH) receptor and a peptide encoded by an RNA that is complementary to ACTH mRNA. Proc. Natl. Acad. Sci. USA 82, 1372-1375. Brentani, R. R., Ribeiro, S. F., Potocnjak, P., Pasqualini, R., Lopes, J. D. and Nakaie, C. R. (1988). Characterization of the cellular receptor for fibronectin through a hydropathic complementarity approach. Proc. Natl. Acad. Si. USA 85. 364-367. Carr, D. J. J., Bost, K. L. and Blalock, J. E. (1986). An antibody to a peptide specified by an RNA that is complementary to gamma-endorphin mRNA recognizes an opiate receptor. J. Neuroirnrnunol. 12, 329-337. Dayhoff, M. 0. and Orcutt, B. C. (1979). Methods for identifying proteins by using partial sequences. Proc. Natl. Acad. Sci. USA 76, 2170-2174. Dayhoff, M. O., Baker, W. C. and Hunt, L. 1. (1983). Establishing homologies in protein sequences. Methods Enzyrnol. 91, 524-545. Elton, T. S., Dion, L. D., Bost, K. L., Oparil, S. and Blalock, J. E. (1988). Purification of an agiotensin II binding protein by using antibodies to a peptide encoded by angiotensin II complementary RNA. Proc. Natl. Acad. Sci. USA 85, 25182522.
Gorcs. T. J., Gottschall. P. E., Coy, D. H. and Arimura, A. (1986). Possible recognition of the GnRH receptor by an antiserum against a peptide encoded by nucleotide sequence complementary to mRNA of a GnRH precursor peptide. feptides 7, 1137-1 145. Johnson, H. M. and Torres, B. A. (1988). A novel arginine vasopressin-binding peptide that blocks arginine vasopressin modulation of immune function. J. lrnrnunol., 141,24202423. Knutson, V. P. (1988). Insulin-binding peptide. J. Biol. Chern. 263, 14146-14151. Kyte, J. and Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J. Mol. Biol. 157, 105-132. Markus, G., Tritsch, G. L. and Parthasarathy, R. (1989). A model for hydropathy-based peptide interactions. Arch. Biochern. Biophys. 272,433-439. Mulchahey, J. J., Neill, J. D., Dion, L. D., Bost, K. L. and Blalock, J. E. (1986). Antibodies to the binding site of the receptor for luteinizing hormone-releasing hormone (LHRH): generation with a synthetic decapeptide encoded by an RNA complementary to LHRH mRNA. Proc. Natl. Acad. Sci. USA 83, 9714-9718. Pascual, D. W., McBurnett, R. T., Blalock, J. E.' and Bost, K. L. (1988). Antigenic relatedness of two peptides specified by the same RNA read in the 5'-3' or 3'-5' direction. FASEB J. 2, 4345. Shai, Y., Flashner, M. and Chaiken, I. M. (1987). Anti-sense peptide recognition of sense peptides: direct quantitative characterization with the ribonuclease S-peptide system using analytical high performance affinity chromatography. Biochemistry 26, 669-675. Soffer, R. L., Bandyopadhyay, S., Rosenberg, E., Hoeprich, P., Teitelbaum, A., Brunck, T., Colby, C. B. and Gloff, C. (1987). Unexpected binding of an octapeptide to the angiotensin I I receptor. Proc. Natl. Acad. Sci. USA 84, 9219-9222. Tritsch, G. L. (1989). Similarity in the hydropathic profile of tyrosyl-t RNA synthetase and the estrogen receptor. FASEB J. 3. 2554.
Proposed Loci of Interaction between Bombesin and its Receptor G. L. Tritsch Department of Experimental Biology, Roswell Park Cancer Institute, New York State Department of Health, Buffalo. 14263. USA
The non-coding strand of the bombesin receptor gene, when ‘translated’ 5’ to 3’, contains an interrupted sequence of the 10 amino acids of bombesin. This finding forms the basis for proposing the points of contact between bombesin and its receptor as well as a partial conformation of the binding region of the receptor.
INTRODUCTION
Recognition in the biological context is thought to involve complementary properties at the interacting surfaces. A remarkable hypothesis has been suggested by Blalock and Smith (1984) based on complementary hydropathy of amino acids of interacting polypeptides. The observation that codons for hydrophobic amino acids of a gene are complemented by codons for hydrophylic amino acids in the non-coding DNA strand, and the converse, is the basis of the hypothesis. The intruiging aspect of this idea is that the genetic code has a role in determining the complementarity of interacting polypeptides. It has further been demonstrated (Tritsch, 1989) that comparable hydropathic profiles of polypeptides suggest a common binding activity. The relevance of this to ligand-receptor interactions was demonstrated by Bost et al. (1985a) by showing that regions of complementarity can be predicted from the gene sequence of epidermal growth factor, interleukin-2, and transferrin with their respective receptors. I n their paper, the non-coding strand of the gene was ‘translated’ 3’ to 5‘ (herein designated nc3’-N with the first amino acid residue as the amino terminus), and in this sequence, several penta- and hexapeptide sequences were found which matched sequences in the respective ligand, albeit not with 100% homology: thus, nucleotide homologies between 53% and 78%, and amino acid homologies of 80% and 83% (one wrong residue in a penta- and hexapeptide sequence, respectively) resulted. Because of redundancy within the genetic code, not all inconsistencies in the gene sequence resulted in changes in the amino acid residues. Other examples of this approach can be found in the literature with constructs prepared nc3’-N (ACTH, Blalock and Boot, 1986), nc3’-C (angiotensin, Soffer et af., 1987), ncS’-N (ACTH, Bost et al., 198%; yendorphin, Carr et ul., 1986; RNAase, Shai etal., 1987; angiotensin, Elton et al., 1988; fibronectin, Brentani et al., 1988; insulin, Knutson, 1988; gonadotrophin releasing hormone, Gorcs et ul., 1986) and nc5‘-C (RNAase. Shai et al., 1987; luteinizing hormone releasing hormone, Mulchahey et al., 1986). Since the gene for the receptor of mammalian bombesin-like peptides has been sequenced recently OY52-.34Y9/Y 1/020053-04 $05 .(HI Wiley & Sons, Ltd
0 19Y1 by John
(Battey ef al., 1991) we derived the non-coding strand from it and examined it for homologous amino acid sequences in bombesin.
RESULTS AND DISCUSSION The bombesin receptor, which is a member of the guanine nucleotide binding protein-coupled receptor superfamily, has an extracellular amino-terminal sequence of 39 amino acids, 7 transmembrane domains, and 1 carboxy-terminal intracellular domain. The entire receptor gene sequence was used to derive the sequence of the non-coding strand, and the latter ‘translated’ from the 5‘ and 3’ ends to obtain the nc5’ and nc3‘ constructs, respectively, as outlined by us previously (Markus et al., 1989). Both of these constructs were examined N to C and C to N to determine if bombesin amino acid sequences could be found. No sequences could be found in the nc3‘ constructs, but the nc5’ construct contained the homologies shown in Fig. 1 in t h e amino-terminal extracellular domain where a total of seven correctly spaced homologies occured with one 5 amino acid space, one 2 amino acid space, and two 1 amino acid spaces. The extracellular domain between membrane-spanning domains 4 and 5 contained three amino acids with a 1 amino acid space that matched three correctly spaced amino acids in bombesin not accounted for by the amino-terminal extracellular domain. Thus, of the 14 amino acids in bombesin, 10 amino acids were found in the receptor by ‘translation’ of the non-coding strand of the gene from the 5‘ end. No homologies could be found in the intracellular or transmembrane portions of the receptor. Because it is highly unlikely that such a long segment be linear in the three-dimensional structure of the bombesinbombesin-receptor complex, the three spaces and the four amino acid residues in bombesin for which no corresponding amino acid residues were found in the construct should not be surprising. Since previous findings of homologies with hexapeptides with one mismatched amino acid were shown to be very highly significant statistically (Bost el af., 198%). finding 10 juxtaposable amino acids constitutes convincing evidence that the sequence of these amino acid residues is highly unlikely to be due to chance. Received 29 March 1991 Accepted (revised) 15 July I991
G . L. TRITSCH
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Figure 1. Genesis of the nc5' construct of the non-coding strand of the bombesin receptor gene. The lines connecting the nucleotides in a given triplet denote canonical Watson-Crick G.C and A.T pairing between the gene and the non-coding strand. The non-coding strand shown was 'translated' 5' to 3' t o produce the amino acids in the nc5' construct. The heavy lines indicate amino acids in the nc5' construct of the gene which are identical to amino acids in bombesin. Note that valine in position 10 of bombesin could be aligned with either of the t w o adjacent valines in the nc5' construct.
If the hydropathic profile of this sequence of 10 amino acids is examined (Fig. 2), it becomes apparent that not all of the 10 juxtaposable amino acids are likely to contribute to binding: interaction between E of bombesin and N of the receptor [both with Kyte and Doolittle (1982) hydropathy scores of - 3.51, between A of bombesin and C of the receptor (hydropathy scores of 1.8 and 2.5, respectively), and between both instances of G of bombesin and T of the receptor (hydropathy scores of - 0.4 and - 0.7, respectively). The hydropathies of these four pairs of amino acids are similar and of the same sign and thus cannot contribute to hydropathic complementarity. When the 10 proposed contact points are used to prepare paired hydropathy plots (Markus et al., 1989) of bombesin and the corresponding amino acids in the receptor (Fig. 3), the results indicate significant hydro-
pathic complementarity ( r = 0.65; slope = 0.67). This level of Complementarity is put into proper perspective when one considers other receptor-ligand interactions. For example, paired hydropathy plots of ACTH and its nc3' and nc5' constructs gave correlation coefficients of 0.79 and 0.61, respectively (Markus et al., 1989). Both of these constructs competed with ACTH in binding experiments in several biological systems with affinity constants in the nanomolar range (Blalock and Bost, 1986). Binding of the nc3' and ncS' constructs of substance P were equivalent (Pascual et al., 1988) as were the correlation coefficients of paired hydropathy plots with substance P ( r = 0 . 8 3 for nc3'; r=0.76 for nc5'). Another example, binding of the nonapeptide vasopressin to its receptor was inhibited only by the nc3' construct; the ncS' construct was inactive (Johnson and Torres, 1988). When the appropriate paired hydro-
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Figure 2. Hydropathic profile of bombesin (H) and its The Kyte and Doolittle (1982) hydropathy scale receptor (a-0).
is used.
Figure 3. Paired hydropathy plot for the 10 amino acids i n bombesin and the amino acids to which they are bound in the receptor (see Fig. 1). The hydropathy scale of Kyte and Doolittle (1982) is used, and the value for an amino acid in the receptor is plotted on the abscissa and the value of the corresponding amino acid i n bombesin is plotted on the ordinate. Negative values to the left and below the origin denote hydrophilic amino acids and positive values, to the right and above the origin, hydrophobic amino acids. The concentric circles indicate that t w o amino acid pairs have identical coordinates. The slope of the least square linear regression shown is -0.67 with a correlation coefficient of 0.65.
PROPOSED INTERACTION LOCI OF BOMBESIN AND ITS RECEPTOR
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Figure 4. Scheme of the bombesin binding domain within the bombesin receptor. (a) The cell membrane is stippled with the extracellular portion on top and the intracellular portion on the bottom. The length of the lines are in proportion to the number of amino acid residues (scale indicated) in each extracellular and intracellular (horizontal lines) domain and transmembrane (vertical lines) domain as deduced from the amino acid sequence (Battey et a/., 1991). In this linear projection, the domains are arranged so that the bombesin binding regions proposed in Fig. 1 can be aligned opposite each other, N-C, to accomodate bombesin, but the lengths of the straight lines which are in direct proportion to the number of amino acids they contain does not allow alignment of the t w o highlighted receptor contact regions as in Fig. 1; the pair of broken lines denotes this displacement. This is obviated in (b). For clarity, bombesin has not been added but is intended t o fit between the t w o highlighted regions in the t w o extracellular domains of the receptor. The transmembrane domains are numbered as i n Battey e t a / . (1991), and 'N' and 'C' denote the amino and carboxy termini of the receptor. It is of interest that the total number of amino acids connecting transmembrane domains 2, 3 and 4 are equal to those connecting transmembrane domains 4 and 5. (b) A view from outside the cell, looking down on the receptor. The extracellular domains are drawn as solid lines and the intracellular domains as broken lines. The same scale as in (a) is used t o denote the number of amino acid residues i n the domains linking the transmembrane domains. Here, the transmembrane domains are arranged in a circle and this allows alignment of the contact points between bombesin and its receptor as shown in Fig. 1. The configuration of the domains between transmembrane domain 5 and the carboxy terminus is arbitrary and unrelated t o the data in Fig. 1.
pathy plots are prepared (data not shown), correlation coefficients of 0.65 and 0.26 for the nc3' and nc5' constructs, respectively, are found. Thus, it becomes apparent that only those constructs which yield amino acid sequences which have correlation coefficients in paired hydropathy plots of ca 0.6 and above should be expected to be active biologically. If the data from the four pairs of amino acids of similar hydropathy of Fig. 2 are removed from consideration (data points in the upper right quadrant and lower left quandrant of Fig. 3 ) . the least square linear regression based on the six remaining juxtaposable amino acid pairs has a slope of - 1 .O1 with a correlation coefficient of 0.997. From this present analysis one would predict that bombesin makes contact with its receptor in the extracellular amino terminal domain as well as with the extracellular domain between the fourth and fifth transmembrane domains (Fig. 4). Here we have complementarity of 10 amino acids. Since the sequence of the bombesin gene is not yet known. we do not know the degree of nucleotide homology which is generally less than amino acid homology due to redundancy within the genetic code. The probability that these amino acid homologies could accur accidentally is unlikely: Dayhoff er a f . (1983) have shown that ' . . . a search for a specific penta- or hexapeptide will usually turn up the source protein and its close relatives or nothing at all'. 'For example, the sequence GDSGG surrounding the serine active site of trypsin is absolutely conserved in all 19 of the sequenced serine proteases related to trypsin, including four bacterial sequences, and it occurs
nowhere else in our present protein sequence data base.' Furthermore, Dayhoff and Orcutt (1979) have stated that 'If a protein sequence is known, a segment can usually be identified with confidence through comparison with the data file of all known sequences when the identity and position of only 7 amino acid residues (not necessarily contiguous) are known'. These findings were confirmed by Bost et al. (1985a) in their analysis of ligand-receptor interactions. The NBRF Protein Information Resource data base ( S O 908 sequence entries) was searched for homologies of the sequence of the bombesin receptor and its nc5' construct which encompasses our proposed loci of interaction (Fig. 1) in both directions, i.e., NLDVDPFLSCNDTFNQ and QNFTDNCSLFPDVDLN. The highest degree of identity found was for 6 out of 16 amino acids (37.5%) as compared to our 10 out of 16 amino acids (62.5%). These lesser homologies were found in rat S-acyl fatty acid synthase thioesterase and rat oleoyl-[acyl-carrierprotein] hydrolase (.LD.. .FL.. .DT...), Escherichia coli NADP-specific glutamate dehydrogenase (NL ...,FL ....TF..) and in mouse and human dihydrofo.FP..DL.). Of all of these, only late reductase (Q.F the dihydrofolate reductase homologies are found when the amino acid sequence was the reverse of that shown in Fig. 1 , i.e., QNFTDNCSLFPDVDLN. The pattern of these homologies differ among themselves as well as from the ones shown in Fig. 1. None of the proteins identified in the search function as receptors, and the positions of homologies are separated by multiple gaps of three or more amino acid residues. Because
Figure 2. Three-dimensional structure of the Ha-ras oncogene product p21 shown as a stereo pair in thea-carbon backbone presentation. The GTP molecule is given in yellow within the active site. The 'hot spots'are identified by the amino acid residuesand Van der Waalssurfaces given in red. All 'hot spots'are clustered in and around the GTP binding active site.
56
G . L. TRITSCH
of this, we did not search the data base for the missing amino acid homologies in other portions of the amino acid sequence of the respective proteins.
amino acids suggests that these regions are involved in the binding of bombesin to its receptor. Crystallographic data, and possibly active site directed mutagenesis studies will confirm or refute this suggestion.
CONCLUSION It is thus concluded that the likelihood that our amino acid homologies could occur accidentally appears to be exceedingly small. This communication is the first demonstration of the use of information derived from the non-coding strand Of a gene Of a sing1e sequence in a ligand binding to non-continuous sequences of a receptor. Thus, finding 10 closely spaced homologous
Acknowledgements The NBRF Protein Information Resource data base search was expertly performed by G . E. Franke, MLS, of the Medical and Scientific Library of this Institute. Appreciation is expressed to Dr Gabor Markus for his valuable discussions during the course of this work and for critical evaluation of this manuscript.
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