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Differential Determinants for Peptide and Non-peptidyl Ligand Binding to the Motilin Receptor

2002; Elsevier BV; Volume: 277; Issue: 12 Linguagem: Inglês

10.1074/jbc.m111051200

1083-351X

Bunzo Matsuura, Maoqing Dong, Laurence J. Miller,

Neuropeptides and Animal Physiology

The predicted second extracellular loop domain of the motilin receptor is of particular interest because it is a region that is quite distinct from the analogous regions in other family members that are most closely related and because the initial report of the photoaffinity labeling of a domain of this receptor included this region (Coulie, B. J., Matsuura, B., Dong, M., Hadac, E. M., Pinon, D. I., Feighner, S. D., Howard, A. D., and Miller, L. J. (2001) J. Biol. Chem. 276, 35518–35522). In the current work, motilin receptor constructs were prepared that included sequential deletions ranging from single residues to twelve amino acid segments throughout this 67 amino acid domain. Each construct was expressed in COS cells and characterized for motilin radioligand binding and motilin-stimulated intracellular calcium responses. The only segments that had negative impact on motilin binding and biological activity included deletion constructs ΔCys235, Δ179–182, and Δ241–246. Cys235is likely involved in the highly conserved and functionally important disulfide bond linking the first and second loops of G protein-coupled receptors. Alanine replacements for each of the amino acid residues in the other two segments revealed that the perimembranous residues at both ends of this loop, Val179 and Leu245 and Arg246, were responsible for the negative impact on motilin binding and biological activity. Of note, these mutants responded normally to the non-peptidyl agonist, erythromycin. These data support important functional roles for both amino-terminal and carboxyl-terminal perimembranous regions of the second loop for responses to the natural agonist peptide, while supporting independent determinants for action of a non-peptidyl agonist ligand. The predicted second extracellular loop domain of the motilin receptor is of particular interest because it is a region that is quite distinct from the analogous regions in other family members that are most closely related and because the initial report of the photoaffinity labeling of a domain of this receptor included this region (Coulie, B. J., Matsuura, B., Dong, M., Hadac, E. M., Pinon, D. I., Feighner, S. D., Howard, A. D., and Miller, L. J. (2001) J. Biol. Chem. 276, 35518–35522). In the current work, motilin receptor constructs were prepared that included sequential deletions ranging from single residues to twelve amino acid segments throughout this 67 amino acid domain. Each construct was expressed in COS cells and characterized for motilin radioligand binding and motilin-stimulated intracellular calcium responses. The only segments that had negative impact on motilin binding and biological activity included deletion constructs ΔCys235, Δ179–182, and Δ241–246. Cys235is likely involved in the highly conserved and functionally important disulfide bond linking the first and second loops of G protein-coupled receptors. Alanine replacements for each of the amino acid residues in the other two segments revealed that the perimembranous residues at both ends of this loop, Val179 and Leu245 and Arg246, were responsible for the negative impact on motilin binding and biological activity. Of note, these mutants responded normally to the non-peptidyl agonist, erythromycin. These data support important functional roles for both amino-terminal and carboxyl-terminal perimembranous regions of the second loop for responses to the natural agonist peptide, while supporting independent determinants for action of a non-peptidyl agonist ligand. [2-(Trimethylammonium) ethyl]methanethiosulfonate bromide high pressure liquid chromatography The superfamily of guanine nucleotide-binding protein (G protein)-coupled receptors is remarkable for its size and diversity, having the largest number of receptors and the greatest variation in natural ligands. These receptors have been organized into classes that share major structural themes, and into smaller groups called families that share a finer level of structural similarities (2Kolakowski L.F. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar). It is noteworthy that receptors in a single family often also have natural ligands that share structural similarities with each other as well. It has been assumed that these ligands bind to the receptors in their family with similar themes.It was recently established that the motilin receptor and growth hormone secretagogue receptors comprise a new family within class I G protein-coupled receptors (3Feighner S.D. Tan C.P. McKee K.K. Palyha O.C. Hreniuk D.L. Pong S.S. Austin C.P. Figueroa D. MacNeil D. Cascieri M.A. Nargund R. Bakshi R. Abramovitz M. Stocco R. Kargman S. O'Neill G. Van der Ploeg L.H. Evans J. Patchett A.A. Smith R.G. Howard A.D. Science. 1999; 284: 2184-2188Crossref PubMed Scopus (344) Google Scholar, 4Howard A.D. Feighner S.D. Cully D.F. Arena J.P. Liberator P.A. Rosenblum C.I. Hamelin M. Hreniuk D.L. Palyha O.C. Anderson J. Paress P.S. Diaz C. Chou M. Liu K.K. McKee K.K. Pong S.S. Chaung L.Y. Elbrecht A. Dashkevicz M. Heavens R. Rigby M. Sirinathsinghji D.J.S. Dean D.C. Melillo D.G. Patchett A.A. Nargund R. Griffin P.R. DeMartino J.A. Gupta S.K. Schaeffer J.M. Smith R.G. Van der Ploeg L.H. Science. 1996; 273: 974-977Crossref PubMed Scopus (1790) Google Scholar, 5McKee K.K. Tan C.P. Palyha O.C. Liu J. Feighner S.D. Hreniuk D.L. Smith R.G. Howard A.D. Van der Ploeg L.H. Genomics. 1997; 46: 426-434Crossref PubMed Scopus (239) Google Scholar). As might be expected, when a natural ligand for the growth hormone secretagogue receptor was subsequently identified as ghrelin, it had significant primary sequence homology with the motilin peptide (6Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7159) Google Scholar, 7Hosoda H. Kojima M. Matsuo H. Kangawa K. J. Biol. Chem. 2000; 275: 21995-22000Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). However, both motilin and ghrelin have unique structural features that make the molecular basis of receptor binding and activation of these receptors particularly interesting.This relates to the requirement in ghrelin for the novel post-translational modification of N-octanoylation of the Ser residue in its third position (6Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7159) Google Scholar). No analogous modification of motilin peptides has been reported. Further, motilin has an unusual structure-activity relationship for a peptide receptor in the class I family of G protein-coupled receptors, having amino-terminal rather than the predominantly carboxyl-terminal sequence determinants for its selectivity of binding and action (3Feighner S.D. Tan C.P. McKee K.K. Palyha O.C. Hreniuk D.L. Pong S.S. Austin C.P. Figueroa D. MacNeil D. Cascieri M.A. Nargund R. Bakshi R. Abramovitz M. Stocco R. Kargman S. O'Neill G. Van der Ploeg L.H. Evans J. Patchett A.A. Smith R.G. Howard A.D. Science. 1999; 284: 2184-2188Crossref PubMed Scopus (344) Google Scholar, 8Poitras P. Gagnon D. St. Pierre S. Biochem. Biophys. Res. Commun. 1992; 183: 36-40Crossref PubMed Scopus (32) Google Scholar).Despite the clear sequence homology between motilin and growth hormone secretagogue receptor sequences, particularly in their predicted transmembrane segments (86%), there is a prominent difference in the region predicted to represent the second extracellular loop domain (Fig. 1). This 67-amino acid loop within the motilin receptor is 40 residues longer than the analogous loop in the growth hormone secretagogue receptor. However, whether these additional residues are functionally significant is not yet clear.In the current work, we focused on the second extracellular loop domain of the motilin receptor and used site-directed mutagenesis with sequential deletion of segments and alanine-scanning mutagenesis to identify functionally important residues. Of note, both ends of this loop, representing domains that are conserved throughout this family, were found to be functionally important for the binding and action of the natural peptide agonist, motilin, whereas the nonconserved residues in the mid-region of the loop were not necessary. Additionally, those regions that were important for peptide responses were not critical for responses to a non-peptidyl agonist known to act at this receptor, erythromycin, supporting distinct receptor structural determinants for action of these two chemically distinct agonist ligands. Although disruption of the conserved disulfide bond that links this extracellular loop to the first extracellular loop domain of this receptor by deletion of a component cysteine (Cys235) completely eliminated calcium signaling in response to the natural peptide agonist, motilin, erythromycin continued to elicit a response in this construct. This finding further supported the distinct structural basis for the action of peptide and non-peptidyl agonist ligands of this receptor.DISCUSSIONThe motilin-growth hormone secretagogue family of receptors may follow unique and interesting molecular themes for binding and being activated by their natural ligands. This relates to the location of critical determinants for binding and biological activity at the amino-terminal ends of both motilin (3Feighner S.D. Tan C.P. McKee K.K. Palyha O.C. Hreniuk D.L. Pong S.S. Austin C.P. Figueroa D. MacNeil D. Cascieri M.A. Nargund R. Bakshi R. Abramovitz M. Stocco R. Kargman S. O'Neill G. Van der Ploeg L.H. Evans J. Patchett A.A. Smith R.G. Howard A.D. Science. 1999; 284: 2184-2188Crossref PubMed Scopus (344) Google Scholar, 8Poitras P. Gagnon D. St. Pierre S. Biochem. Biophys. Res. Commun. 1992; 183: 36-40Crossref PubMed Scopus (32) Google Scholar) and ghrelin (6Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7159) Google Scholar) rather than at the carboxyl terminus, which is most typical of peptide receptors within the class I G protein-coupled receptors (2Kolakowski L.F. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar). It is also possible that the members of this receptor family follow themes that are distinct even among themselves. This relates to the novel and functionally critical post-translational modification of ghrelin, N-octanoylation of Ser3 (6Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7159) Google Scholar), which is apparently absent in motilin. Such a modification could establish an association with the lipid bilayer that orients ghrelin in a unique manner prior to its receptor binding.To explore the possibility of distinct modes of binding for members of this receptor family, we focused our efforts on a domain of substantial difference between the motilin and growth hormone secretagogue receptors in the second extracellular loop domain. This region includes a 40-amino acid insertion in the motilin receptor that is absent in the growth hormone secretagogue receptor. The potential importance of this unique region was further emphasized by the recent report of the first photoaffinity labeling of a motilin receptor domain that included this loop (1Coulie B.J. Matsuura B. Dong M. Hadac E.M. Pinon D.I. Feighner S.D. Howard A.D. Miller L.J. J. Biol. Chem. 2001; 276: 35518-35522Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar).We followed the classical experimental approaches of deletional and alanine-scanning mutagenesis. The results of these efforts focused interest on three regions within the second extracellular loop. A key amino acid is Cys235, a highly conserved residue that contributes to a conserved disulfide bond linking the first and second extracellular loop domains of essentially every member of the G protein-coupled receptor superfamily. Deletions on both sides of this residue had no negative impact, whereas deletion of only this residue had a profound negative effect on receptor biosynthesis and its ability to bind and signal in response to motilin. Even micromolar concentrations of motilin elicited no intracellular calcium response from cells expressing this construct. In contrast, and of particular interest, cells expressing this construct were still able to respond to the non-peptidyl agonist, erythromycin. This represented a clear indication that non-peptidyl agonist had receptor structural determinants for binding and action that were clearly distinct from those for the natural peptide agonist ligand.The second region of interest represented the amino-terminal perimembranous domain of the second loop. Here, Val179 was shown to be responsible for the negative impact of the mutants on motilin responses. There was evidence that the V179A construct is synthesized normally and undergoes normal trafficking to the cell surface, because there was a small biological response to the cell-impermeant peptide ligand, motilin, even though its affinity was too low to detect by radioligand binding assay. This negative impact on motilin binding and biological activity could be either direct, interacting with the bound peptide ligand, or indirect, acting via an allosteric effect on receptor conformation. Because this hydrophobic amino acid is predicted to reside at or near the interface with the lipid bilayer, many potentially important allosteric effects can be proposed. An important control demonstrated normal responsiveness to erythromycin. This likely reflects the different receptor domains necessary for the action of peptide and non-peptidyl agonist ligands.The third region of interest represented the carboxyl-terminal perimembranous domain of the second loop. Here, both Leu245 and Arg246 appear to contribute to the negative impact of the mutants. Here, too, there was adequate evidence for normal biosynthesis and trafficking to the cell surface, where both binding and signaling were markedly affected. This negative effect may also be either direct or indirect. Here, too, the non-peptidyl agonist, erythromycin, elicited entirely normal biological responses.The same types of considerations described above for the amino-terminal perimembranous domain are relevant here as well. Both of these domains would be expected to be spatially approximated with each other in the intact, fully folded receptor molecule. At the present time, there is no credible model to illustrate this, although the constraint contributed by the current support for the disulfide bond linking the first and second loop domains will provide a starting point for developing such a model.These observations indeed support the functional importance of the motilin receptor second extracellular loop region for binding and activation by the natural peptide ligand. Perhaps surprisingly, the structural feature of this loop that was most responsible for focusing our attention in these studies, the 40-amino acid residue insertion not present in the growth hormone secretagogue receptor, appears to play no functional role. Instead, the functionally important domains within this extracellular loop are all conserved in the other structurally related G protein-coupled receptors. These include the highly conserved disulfide bond that almost certainly has a global architectural role in the superfamily and also both extreme perimembranous regions of this loop. The precise functional roles for peptide binding and action for the identified key residues remain to be defined. The superfamily of guanine nucleotide-binding protein (G protein)-coupled receptors is remarkable for its size and diversity, having the largest number of receptors and the greatest variation in natural ligands. These receptors have been organized into classes that share major structural themes, and into smaller groups called families that share a finer level of structural similarities (2Kolakowski L.F. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar). It is noteworthy that receptors in a single family often also have natural ligands that share structural similarities with each other as well. It has been assumed that these ligands bind to the receptors in their family with similar themes. It was recently established that the motilin receptor and growth hormone secretagogue receptors comprise a new family within class I G protein-coupled receptors (3Feighner S.D. Tan C.P. McKee K.K. Palyha O.C. Hreniuk D.L. Pong S.S. Austin C.P. Figueroa D. MacNeil D. Cascieri M.A. Nargund R. Bakshi R. Abramovitz M. Stocco R. Kargman S. O'Neill G. Van der Ploeg L.H. Evans J. Patchett A.A. Smith R.G. Howard A.D. Science. 1999; 284: 2184-2188Crossref PubMed Scopus (344) Google Scholar, 4Howard A.D. Feighner S.D. Cully D.F. Arena J.P. Liberator P.A. Rosenblum C.I. Hamelin M. Hreniuk D.L. Palyha O.C. Anderson J. Paress P.S. Diaz C. Chou M. Liu K.K. McKee K.K. Pong S.S. Chaung L.Y. Elbrecht A. Dashkevicz M. Heavens R. Rigby M. Sirinathsinghji D.J.S. Dean D.C. Melillo D.G. Patchett A.A. Nargund R. Griffin P.R. DeMartino J.A. Gupta S.K. Schaeffer J.M. Smith R.G. Van der Ploeg L.H. Science. 1996; 273: 974-977Crossref PubMed Scopus (1790) Google Scholar, 5McKee K.K. Tan C.P. Palyha O.C. Liu J. Feighner S.D. Hreniuk D.L. Smith R.G. Howard A.D. Van der Ploeg L.H. Genomics. 1997; 46: 426-434Crossref PubMed Scopus (239) Google Scholar). As might be expected, when a natural ligand for the growth hormone secretagogue receptor was subsequently identified as ghrelin, it had significant primary sequence homology with the motilin peptide (6Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7159) Google Scholar, 7Hosoda H. Kojima M. Matsuo H. Kangawa K. J. Biol. Chem. 2000; 275: 21995-22000Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). However, both motilin and ghrelin have unique structural features that make the molecular basis of receptor binding and activation of these receptors particularly interesting. This relates to the requirement in ghrelin for the novel post-translational modification of N-octanoylation of the Ser residue in its third position (6Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7159) Google Scholar). No analogous modification of motilin peptides has been reported. Further, motilin has an unusual structure-activity relationship for a peptide receptor in the class I family of G protein-coupled receptors, having amino-terminal rather than the predominantly carboxyl-terminal sequence determinants for its selectivity of binding and action (3Feighner S.D. Tan C.P. McKee K.K. Palyha O.C. Hreniuk D.L. Pong S.S. Austin C.P. Figueroa D. MacNeil D. Cascieri M.A. Nargund R. Bakshi R. Abramovitz M. Stocco R. Kargman S. O'Neill G. Van der Ploeg L.H. Evans J. Patchett A.A. Smith R.G. Howard A.D. Science. 1999; 284: 2184-2188Crossref PubMed Scopus (344) Google Scholar, 8Poitras P. Gagnon D. St. Pierre S. Biochem. Biophys. Res. Commun. 1992; 183: 36-40Crossref PubMed Scopus (32) Google Scholar). Despite the clear sequence homology between motilin and growth hormone secretagogue receptor sequences, particularly in their predicted transmembrane segments (86%), there is a prominent difference in the region predicted to represent the second extracellular loop domain (Fig. 1). This 67-amino acid loop within the motilin receptor is 40 residues longer than the analogous loop in the growth hormone secretagogue receptor. However, whether these additional residues are functionally significant is not yet clear. In the current work, we focused on the second extracellular loop domain of the motilin receptor and used site-directed mutagenesis with sequential deletion of segments and alanine-scanning mutagenesis to identify functionally important residues. Of note, both ends of this loop, representing domains that are conserved throughout this family, were found to be functionally important for the binding and action of the natural peptide agonist, motilin, whereas the nonconserved residues in the mid-region of the loop were not necessary. Additionally, those regions that were important for peptide responses were not critical for responses to a non-peptidyl agonist known to act at this receptor, erythromycin, supporting distinct receptor structural determinants for action of these two chemically distinct agonist ligands. Although disruption of the conserved disulfide bond that links this extracellular loop to the first extracellular loop domain of this receptor by deletion of a component cysteine (Cys235) completely eliminated calcium signaling in response to the natural peptide agonist, motilin, erythromycin continued to elicit a response in this construct. This finding further supported the distinct structural basis for the action of peptide and non-peptidyl agonist ligands of this receptor. DISCUSSIONThe motilin-growth hormone secretagogue family of receptors may follow unique and interesting molecular themes for binding and being activated by their natural ligands. This relates to the location of critical determinants for binding and biological activity at the amino-terminal ends of both motilin (3Feighner S.D. Tan C.P. McKee K.K. Palyha O.C. Hreniuk D.L. Pong S.S. Austin C.P. Figueroa D. MacNeil D. Cascieri M.A. Nargund R. Bakshi R. Abramovitz M. Stocco R. Kargman S. O'Neill G. Van der Ploeg L.H. Evans J. Patchett A.A. Smith R.G. Howard A.D. Science. 1999; 284: 2184-2188Crossref PubMed Scopus (344) Google Scholar, 8Poitras P. Gagnon D. St. Pierre S. Biochem. Biophys. Res. Commun. 1992; 183: 36-40Crossref PubMed Scopus (32) Google Scholar) and ghrelin (6Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7159) Google Scholar) rather than at the carboxyl terminus, which is most typical of peptide receptors within the class I G protein-coupled receptors (2Kolakowski L.F. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar). It is also possible that the members of this receptor family follow themes that are distinct even among themselves. This relates to the novel and functionally critical post-translational modification of ghrelin, N-octanoylation of Ser3 (6Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7159) Google Scholar), which is apparently absent in motilin. Such a modification could establish an association with the lipid bilayer that orients ghrelin in a unique manner prior to its receptor binding.To explore the possibility of distinct modes of binding for members of this receptor family, we focused our efforts on a domain of substantial difference between the motilin and growth hormone secretagogue receptors in the second extracellular loop domain. This region includes a 40-amino acid insertion in the motilin receptor that is absent in the growth hormone secretagogue receptor. The potential importance of this unique region was further emphasized by the recent report of the first photoaffinity labeling of a motilin receptor domain that included this loop (1Coulie B.J. Matsuura B. Dong M. Hadac E.M. Pinon D.I. Feighner S.D. Howard A.D. Miller L.J. J. Biol. Chem. 2001; 276: 35518-35522Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar).We followed the classical experimental approaches of deletional and alanine-scanning mutagenesis. The results of these efforts focused interest on three regions within the second extracellular loop. A key amino acid is Cys235, a highly conserved residue that contributes to a conserved disulfide bond linking the first and second extracellular loop domains of essentially every member of the G protein-coupled receptor superfamily. Deletions on both sides of this residue had no negative impact, whereas deletion of only this residue had a profound negative effect on receptor biosynthesis and its ability to bind and signal in response to motilin. Even micromolar concentrations of motilin elicited no intracellular calcium response from cells expressing this construct. In contrast, and of particular interest, cells expressing this construct were still able to respond to the non-peptidyl agonist, erythromycin. This represented a clear indication that non-peptidyl agonist had receptor structural determinants for binding and action that were clearly distinct from those for the natural peptide agonist ligand.The second region of interest represented the amino-terminal perimembranous domain of the second loop. Here, Val179 was shown to be responsible for the negative impact of the mutants on motilin responses. There was evidence that the V179A construct is synthesized normally and undergoes normal trafficking to the cell surface, because there was a small biological response to the cell-impermeant peptide ligand, motilin, even though its affinity was too low to detect by radioligand binding assay. This negative impact on motilin binding and biological activity could be either direct, interacting with the bound peptide ligand, or indirect, acting via an allosteric effect on receptor conformation. Because this hydrophobic amino acid is predicted to reside at or near the interface with the lipid bilayer, many potentially important allosteric effects can be proposed. An important control demonstrated normal responsiveness to erythromycin. This likely reflects the different receptor domains necessary for the action of peptide and non-peptidyl agonist ligands.The third region of interest represented the carboxyl-terminal perimembranous domain of the second loop. Here, both Leu245 and Arg246 appear to contribute to the negative impact of the mutants. Here, too, there was adequate evidence for normal biosynthesis and trafficking to the cell surface, where both binding and signaling were markedly affected. This negative effect may also be either direct or indirect. Here, too, the non-peptidyl agonist, erythromycin, elicited entirely normal biological responses.The same types of considerations described above for the amino-terminal perimembranous domain are relevant here as well. Both of these domains would be expected to be spatially approximated with each other in the intact, fully folded receptor molecule. At the present time, there is no credible model to illustrate this, although the constraint contributed by the current support for the disulfide bond linking the first and second loop domains will provide a starting point for developing such a model.These observations indeed support the functional importance of the motilin receptor second extracellular loop region for binding and activation by the natural peptide ligand. Perhaps surprisingly, the structural feature of this loop that was most responsible for focusing our attention in these studies, the 40-amino acid residue insertion not present in the growth hormone secretagogue receptor, appears to play no functional role. Instead, the functionally important domains within this extracellular loop are all conserved in the other structurally related G protein-coupled receptors. These include the highly conserved disulfide bond that almost certainly has a global architectural role in the superfamily and also both extreme perimembranous regions of this loop. The precise functional roles for peptide binding and action for the identified key residues remain to be defined. The motilin-growth hormone secretagogue family of receptors may follow unique and interesting molecular themes for binding and being activated by their natural ligands. This relates to the location of critical determinants for binding and biological activity at the amino-terminal ends of both motilin (3Feighner S.D. Tan C.P. McKee K.K. Palyha O.C. Hreniuk D.L. Pong S.S. Austin C.P. Figueroa D. MacNeil D. Cascieri M.A. Nargund R. Bakshi R. Abramovitz M. Stocco R. Kargman S. O'Neill G. Van der Ploeg L.H. Evans J. Patchett A.A. Smith R.G. Howard A.D. Science. 1999; 284: 2184-2188Crossref PubMed Scopus (344) Google Scholar, 8Poitras P. Gagnon D. St. Pierre S. Biochem. Biophys. Res. Commun. 1992; 183: 36-40Crossref PubMed Scopus (32) Google Scholar) and ghrelin (6Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7159) Google Scholar) rather than at the carboxyl terminus, which is most typical of peptide receptors within the class I G protein-coupled receptors (2Kolakowski L.F. Receptors Channels. 1994; 2: 1-7PubMed Google Scholar). It is also possible that the members of this receptor family follow themes that are distinct even among themselves. This relates to the novel and functionally critical post-translational modification of ghrelin, N-octanoylation of Ser3 (6Kojima M. Hosoda H. Date Y. Nakazato M. Matsuo H. Kangawa K. Nature. 1999; 402: 656-660Crossref PubMed Scopus (7159) Google Scholar), which is apparently absent in motilin. Such a modification could establish an association with the lipid bilayer that orients ghrelin in a unique manner prior to its receptor binding. To explore the possibility of distinct modes of binding for members of this receptor family, we focused our efforts on a domain of substantial difference between the motilin and growth hormone secretagogue receptors in the second extracellular loop domain. This region includes a 40-amino acid insertion in the motilin receptor that is absent in the growth hormone secretagogue receptor. The potential importance of this unique region was further emphasized by the recent report of the first photoaffinity labeling of a motilin receptor domain that included this loop (1Coulie B.J. Matsuura B. Dong M. Hadac E.M. Pinon D.I. Feighner S.D. Howard A.D. Miller L.J. J. Biol. Chem. 2001; 276: 35518-35522Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). We followed the classical experimental approaches of deletional and alanine-scanning mutagenesis. The results of these efforts focused interest on three regions within the second extracellular loop. A key amino acid is Cys235, a highly conserved residue that contributes to a conserved disulfide bond linking the first and second extracellular loop domains of essentially every member of the G protein-coupled receptor superfamily. Deletions on both sides of this residue had no negative impact, whereas deletion of only this residue had a profound negative effect on receptor biosynthesis and its ability to bind and signal in response to motilin. Even micromolar concentrations of motilin elicited no intracellular calcium response from cells expressing this construct. In contrast, and of particular interest, cells expressing this construct were still able to respond to the non-peptidyl agonist, erythromycin. This represented a clear indication that non-peptidyl agonist had receptor structural determinants for binding and action that were clearly distinct from those for the natural peptide agonist ligand. The second region of interest represented the amino-terminal perimembranous domain of the second loop. Here, Val179 was shown to be responsible for the negative impact of the mutants on motilin responses. There was evidence that the V179A construct is synthesized normally and undergoes normal trafficking to the cell surface, because there was a small biological response to the cell-impermeant peptide ligand, motilin, even though its affinity was too low to detect by radioligand binding assay. This negative impact on motilin binding and biological activity could be either direct, interacting with the bound peptide ligand, or indirect, acting via an allosteric effect on receptor conformation. Because this hydrophobic amino acid is predicted to reside at or near the interface with the lipid bilayer, many potentially important allosteric effects can be proposed. An important control demonstrated normal responsiveness to erythromycin. This likely reflects the different receptor domains necessary for the action of peptide and non-peptidyl agonist ligands. The third region of interest represented the carboxyl-terminal perimembranous domain of the second loop. Here, both Leu245 and Arg246 appear to contribute to the negative impact of the mutants. Here, too, there was adequate evidence for normal biosynthesis and trafficking to the cell surface, where both binding and signaling were markedly affected. This negative effect may also be either direct or indirect. Here, too, the non-peptidyl agonist, erythromycin, elicited entirely normal biological responses. The same types of considerations described above for the amino-terminal perimembranous domain are relevant here as well. Both of these domains would be expected to be spatially approximated with each other in the intact, fully folded receptor molecule. At the present time, there is no credible model to illustrate this, although the constraint contributed by the current support for the disulfide bond linking the first and second loop domains will provide a starting point for developing such a model. These observations indeed support the functional importance of the motilin receptor second extracellular loop region for binding and activation by the natural peptide ligand. Perhaps surprisingly, the structural feature of this loop that was most responsible for focusing our attention in these studies, the 40-amino acid residue insertion not present in the growth hormone secretagogue receptor, appears to play no functional role. Instead, the functionally important domains within this extracellular loop are all conserved in the other structurally related G protein-coupled receptors. These include the highly conserved disulfide bond that almost certainly has a global architectural role in the superfamily and also both extreme perimembranous regions of this loop. The precise functional roles for peptide binding and action for the identified key residues remain to be defined. We acknowledge the excellent technical assistance of E. Holicky, D. I. Pinon, and E. M. Hadac, as well as the excellent assistance of E. M. Hadac in the preparation of graphics.