Further definition of the substance P (SP)/neurokinin-1 receptor complex. MET-174 is the site of photoinsertion p-benzoylphenylalanine4 SP

The covalent attachment site of a substance P (SP) analogue containing the photoreactive amino acid p-benzoyl-l-phenylalanine (Bpa) in position 8 of the C-terminal portion of the peptide was identified previously as Met-181 on the neurokinin-1 (NK-1) receptor. In this study, a second photoreactive SP analogue, Bpa(4)-SP, in which the Bpa residue is located in the N-terminal portion of the peptide, was used to define further the peptide-receptor interface. The NK-1 receptor expressed in Chinese hamster ovary cells was specifically and efficiently photolabeled with a radioiodinated derivative of Bpa(4)-SP. Fragmentation analysis of the photolabeled receptor restricted the site of photoincorporation of Bpa(4)-SP to an amino acid within the sequence Thr-173 to Arg-177 located on the N-terminal side of the E2 loop. To identify the specific amino acid in this sequence that serves as the covalent attachment site for Bpa(4)-SP, a small photolabeled receptor fragment was generated by chemical cleavage with cyanogen bromide. Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the purified fragment identified a single protonated molecular ion with a molecular mass of 1801.3 +/- 1.8, indicating that upon irradiation, the bound photoligand covalently attaches to the terminal methyl group of a methionine residue. This result, taken together with the results of the peptide mapping studies, establishes that the site of Bpa(4)-SP covalent attachment to the NK-1 receptor is Met-174.     

Evidence for spatial proximity of two distinct receptor regions in the substance P (SP)*neurokinin-1 receptor (NK-1R) complex obtained by photolabeling the NK-1R with p-benzoylphenylalanine3-SP

Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that (125)I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine(8)-SP (Bpa(8)SP) covalently attaches to Met(181), whereas (125)I-Bolton-Hunter reagent-labeled Bpa(4)SP covalently attaches to Met(174), both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue (125)I-[D-Tyr(0)]Bpa(3)SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation. Since competition binding data presented in this study do not suggest the existence of multiple peptide.NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.     

Molecular characterization of the substance P*neurokinin-1 receptor complex: development of an experimentally based model

Molecular models for the interaction of substance P (SP) with its G protein-coupled receptor, the neurokinin-1 receptor (NK-1R), have been developed. The ligand.receptor complex is based on experimental data from a series of photoaffinity labeling experiments and spectroscopic structural studies of extracellular domains of the NK-1R. Using the ligand/receptor contact points derived from incorporation of photolabile probes (p-benzoylphenylalanine (Bpa)) into SP at positions 3, 4, and 8 and molecular dynamics simulations, the topological arrangement of SP within the NK-1R is explored. The model incorporates the structural features, determined by high resolution NMR studies, of the second extracellular loop (EC2), containing contact points Met(174) and Met(181), providing important experimentally based conformational preferences for the simulations. Extensive molecular dynamics simulations were carried out to probe the nature of the two contact points identified for the Bpa(3)SP analogue (Bremer, A. A., Leeman, S. E., and Boyd, N. D. (2001) J. Biol. Chem. 276, 22857-22861), examining modes of ligand binding in which the contact points are fulfilled sequentially or simultaneously. The resulting ligand.receptor complex has the N terminus of SP projecting toward transmembrane helix (TM) 1 and TM2, exposed to the solvent. The C terminus of SP is located in proximity to TM5 and TM6, deeper into the central core of the receptor. The central portion of the ligand, adopting a helical loop conformation, is found to align with the helices of the central regions EC2 and EC3, forming important interactions with both of these extracellular domains. The model developed here allows for atomic insight into the biochemical data currently available and guides targeting of future experiments to probe specific ligand/receptor interactions and thereby furthers our understanding of the functioning of this important neuropeptide system.     

Met174 side chain is the site of photoinsertion of a substance P competitive peptide antagonist photoreactive in position 8

Numerous photoaffinity studies of the NK-1 receptor have been carried out with peptide agonist analogues of substance P (SP). However, no information is available with regard to the domain interaction of peptide antagonists within this receptor. We describe herein the photoaffinity labelling of the SP receptor with a peptide antagonist analogue, Bapa(0)[(pBzl)Phe(8),DPro(9),MePhe(10),Trp(CHO)(11)]SP. Photolabelling, enzymatic or chemical cleavage of the covalent complex, purification via streptavidin-coated beads and matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis led us to show that the methyl of Met174 side chain, within the receptor's second extracellular loop, is covalently linked to the antagonist photoreactive at position 8.     

Involvement of the second extracellular loop (E2) of the neurokinin-1 receptor in the binding of substance P. Photoaffinity labeling and modeling studies

Substance P (SP) interacts with the neurokinin-1 (NK-1) G-protein-coupled receptor, which has been cloned in several species. In the present study, the domains of the NK-1 receptor involved in the binding of SP and SP-(7-11) C-terminal fragment have been analyzed using two peptide analogs containing the photoreactive amino acid para-benzoylphenylalanine ((p-Bz)Phe) in position 8 of their sequence. This study was carried out with [BAPA-Lys(6),(p-Bz)Phe(8),Pro(9),Met(O(2))(11)]SP-(7-11) and [BAPA(0),(p-Bz)Phe(8)]SP on both rat and human NK-1 receptors expressed in CHO cells. Combined trypsin and endo-GluC enzymatic complete digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis led to the identification of the same domain of covalent interaction, (173)TMPSR(177), for the two photoactivatable peptides. Further digestion of this fragment with carboxypeptidase Y led to the identification of (173)TMP(175) in the second extracellular loop (E2) of the NK-1 receptor as the site of covalent attachment. Models of the conformation of this E2 loop in the human NK-1 receptor were generated using two different strategies, one based on homology with bovine rhodopsin and the other based on the solution conformation preferences of a synthetic peptide corresponding to the E2 loop.     

Cgamma H2 of Met174 side chain is the site of covalent attachment of a substance P analog photoactivable in position 5

Analogs of substance P (H-RPKPQQFFGLM-NH(2)) incorporating a photoreactive para-benzoyl-l-phenylalanine (p-Bzl)Phe at position 4, 5, 6, 9, or 10 of the sequence have been synthesized and pharmacologically characterized previously as full NK-1 receptor agonists. In this study we show that all analogs, [BAPA(0), (p-Bzl)Phe(x), Met(O(2))(11)]SP also display high yields (40-70%) of NK-1 receptor photolabeling. To identify the site of photoinsertion in the receptor, covalent ligand/receptor complexes were digested with enzymes or chemically cleaved with cyanogen bromide and purified with streptavidin-coated magnetic beads before matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. Only the analog photoreactive at position 5 gave irreversible, reproducible, and unequivocal covalent linkage. Sequential digestions of the covalent complex, substance P analog photoreactive at position 5/NK-1 receptor, with trypsin, endo-GluC and carboxypeptidase Y, led to the identification of the tripeptide (173)TMP(175) in the second extracellular loop of the hNK-1 receptor as the site of photoinsertion. Reaction of cyanogen bromide on the pentapeptide TMPSR did not yield the expected cleavage on the carboxylic side of methionine. The high precision of mass spectrometry analysis on the mass measured led us to determine that C(gamma)H(2) of Met(174) was the site of covalent linkage of the photoreactive substance P analog. Such an insertion (photolinked ligand) on its C(gamma)H(2) renders methionine refractory to CNBr cleavage.     

A high-affinity,radioiodinatable neuropeptide FF analogue incorporating a photolabile p-(4-hydroxybenzoyl)phenylalanine

A new radioiodinated photoaffinity compound, [¹²⁵I]YE(Bpa)WSLAAPQRFNH₂, derived from a peptide present in the rat neuropeptide FF (NPFF) precursor was synthesized, and its binding characteristics were investigated on a neuroblastoma clone, SH-SY5Y, stably expressing rat NPFF₂ receptors tagged with the T7 epitope. The binding of the probe was saturable and revealed a high-affinity interaction (K_D = 0.24 nM) with a single class of binding sites. It was also able to affinity label NPFF₂ receptor in a specific and efficient manner given that 38% of the bound radioligand at saturating concentration formed a wash-resistant binding after ultraviolet (UV) irradiation. Photoaffinity labeling with [¹²⁵I]YE(Bpa)WSLAAPQRFamide showed two molecular forms of NPFF₂ receptor with apparent molecular weights of 140 and 95 kDa in a 2:1 ratio. The comparison of the results between photoaffinity labeling and Western blot analysis suggests that all receptor forms bind the probe irreversibly with the same efficiency. On membranes of mouse olfactory bulb, only the high molecular weight form of NPFF₂ receptor is observed. [¹²⁵I]YE(Bpa)WSLAAPQRFamide is an excellent radioiodinated peptidic ligand for direct and selective labeling of NPFF₂ receptors in vitro.     

The common C-terminal sequences of substance P and neurokinin A contact the same region of the NK-1 receptor

Although neurokinin A (NKA), a tachykinin peptide with sequence homology to substance P (SP), is a weak competitor of radiolabeled SP binding to the NK-1 receptor (NK-1R), more recent direct binding studies using radiolabeled NKA have demonstrated an unexpected high-affinity interaction with this receptor. To document the site of interaction between NKA and the NK-1R, we have used a photoreactive analogue of NKA containing p-benzoyl-L-phenylalanine (Bpa) substituted in position 7 of the peptide. Peptide mapping studies of the receptor photolabeled by (125)I-iodohistidyl(1)-Bpa(7)NKA have established that the site of photoinsertion is located within a segment of the receptor extending from residues 178 to 190 (VVCMIEWPEHPNR). We have previously shown that (125)I-BH-Bpa(8)SP, a photoreactive analogue of SP, covalently attaches to M(181) within this same receptor sequence. Importantly, both of these peptides ((125)I-iodohistidyl(1)-Bpa(7)NKA and (125)I-BH-Bpa(8)SP) have the photoreactive amino acid in an equivalent position within the conserved tachykinin carboxyl-terminal tail. In this report, we also show that site-directed mutagenesis of M(181) to A(181) in the NK-1R results in a complete loss of photolabeling of both peptides to this receptor site, indicating that the equivalent position of SP and NKA, when bound to the NK-1R, contact the same residue.     

Photolabeling identifies position 172 of the human AT(1) receptor as a ligand contact point: receptor-bound angiotensin II adopts an extended structure

An angiotensin II (AngII) peptidic analogue in which the third residue (valine) was substituted with the photoreactive p-benzoyl-L-phenylalanine (Bpa) was used to identify ligand-binding sites of the human AT(1) receptor. High-affinity binding of the analogue, (125)I-[Bpa(3)]AngII, to the AT(1) receptor heterologously expressed in COS-7 cells enabled us to efficiently photolabel the receptor. Chemical and enzymatic digestions of the (125)I-[Bpa(3)]AngII-AT(1) complex were performed, and receptor fragments were analyzed in order to define the region of the receptor with which the ligand interacts. Results show that CNBr hydrolysis of the photolabeled receptor gave a glycosylated fragment which, after PNGase-F digestion, migrated as a 11.4 kDa fragment, circumscribing the labeled domain between residues 143-243 of the AT(1) receptor. Digestion of the receptor-ligand complex with Endo Lys-C or trypsin followed by PNGase-F treatment yielded fragments of 7 and 4 kDa, defining the labeling site of (125)I-[Bpa(3)]AngII within residues 168-199 of the AT(1) receptor. Photolabeling of three mutant receptors in which selected residues adjacent to residue 168 were replaced by methionine within the 168-199 fragment (I172M, T175M, and I177M) followed by CNBr cleavage revealed that the bound photoligand (125)I-[Bpa(3)]AngII forms a covalent bond with the side chain of Met(172) of the second extracellular loop of the AT(1) receptor. These data coupled with previously obtained results enable us to propose a model whereby AngII adopts an extended beta-strand conformation when bound to the receptor and would orient itself within the binding domain by having its N-terminal portion interacting with the second extracellular loop and its C-terminus interacting with residues of the seventh transmembrane domain.     

Direct identification of a peptide binding region in the opioid receptor-like 1 receptor by photoaffinity labeling with [Bpa(10),Tyr(14)]nociceptin

The heptadecapeptide nociceptin, also known as orphanin FQ, is the endogenous agonist of the opioid receptor-like 1 (ORL1) G protein-coupled receptor. An affinity labeling approach has been implemented to probe the interactions of the neuropeptide with the receptor using the photolabile nociceptin derivative, [p-benzoyl-l-Phe(10),Tyr(14)]nociceptin ([Bpa(10),Tyr(14)]noc). In recombinant Chinese hamster ovary cells expressing the human ORL1 receptor, [Bpa(10),Tyr(14)]noc binds the receptor with high affinity (K(i) approximately 0.7 nm) and is as potent as nociceptin in the inhibition of forskolin-induced cAMP synthesis (EC(50) approximately 0.5 nm). UV irradiation at 365 nm of the complex formed by the ORL1 receptor and radioiodinated [Bpa(10),Tyr(14)]noc results in the irreversible labeling of a glycoprotein of approximately 65 kDa, determined by SDS-polyacrylamide gel electrophoresis. Complete digestion of the partially purified 65-kDa complex with kallikrein generates a single labeled fragment (approximately 6.5 kDa) that is readily cleaved by endoproteinase Glu-C to yield a labeled fragment of approximately 3.2 kDa. Kallikrein treatment of the photoaffinity cross-linked Glu(295) --> Asp mutant receptor also yields a single labeled fragment of approximately 6.5 kDa but is resistant to further cleavage by endoproteinase Glu-C. Based upon the expected proteolytic fingerprint of the labeled receptor, the photoreactive region can be identified as ORL1-(296-302; residues Thr-Ala-Val-Ala-Ile-Leu-Arg) spanning the C terminus of extracellular loop 3 and the N terminus of transmembrane helix VII. Molecular modeling of the ORL1 receptor complex with [Bpa(10)]noc suggests that reaction of the Bpa carbonyl group may occur with the side chain of Ile(300) within the experimentally identified photoreactive region.     

Apparent molecular weight of substance P/neurokinin-1 receptors determined using a photoaffinity labelled probe, [(3'-125I) D-Tyro, (4'-N3)Phe8, Nle11]-substance P

A photoreactive derivative, [(3'-125I) D-Tyro, (4'-N3)Phe8, Nle11]-substance P (SP) was prepared and iodinated using carrier-free [125I] to determine the apparent molecular weight of one sub-type of neurokinin (NK) receptor, the SP/NK-1 type. The unlabelled analogue competed for [3H]-SP sites with an IC50 of 10 nM. The radioactive photoprobe (KD approximately 0.17 nM, Bmax = 15.6 fmol/mg protein) was used to photoaffinity label membranes prepared from rat brain. Autoradiographs revealed that a single band with an apparent molecular weight of 46,000 daltons was specifically labelled. This labelling was inhibited by non-radioactive SP in a concentration-dependent manner (1.0 nM-0.1 mM) suggesting that the observed labelling represents the SP/NK-1 receptor type.     

Extracellular domains of the neurokinin-1 receptor: structural characterization and interactions with substance P

The technical difficulties associated with the structure determination of membrane proteins have limited the structural information available for the ligand binding to G-protein coupled receptors (GPCRs). Here, we describe a reductionist approach to GPCR structure determination in which the extracellular domains of the receptor are examined by high-resolution NMR in the presence of a membrane mimetic. The resulting structural features are then incorporated into a molecular model of the receptor, utilizing the x-ray structure of rhodopsin to generate the topological orientation of the transmembrane helices. The results of our study of the neurokinin-1 receptor (NK-1R) and its interactions with substance P (SP) are detailed here. The structure of the N-terminus, NK-1R(1-39), and of the third extracellular loop, NK-1R(264-290), in the presence of dodecylphosphocholine micelles is described. Our findings provide a structural basis for the interpretation of the results from other methods including mutagenesis, fluorescence, and photoaffinity labeling experiments, resulting in an experimentally based, high-resolution model of SP binding to NK-1R.     

Synthesis of a potent agonist of substance P by modifying the methionyl and glutaminyl residues of the C-terminal hexapeptide of substance P. Structure-activity relationships.

Analogues of [Orn6]-SP6-11 have been synthesized in which the Met11 residue is replaced by glutamate gamma-alkylesters. These analogues were tested in three in vitro preparations representative of NK-1, NK-2, and NK-3 receptor types. Substitution of the SCH3 group of the Met11 side chain by a COOR (R = methyl, ethyl, n-propyl, n-butyl, cyclohexyl) group results in analogues which are full agonists in NK-1 and NK-2 preparations but show little agonist activity in the NK-3 preparation. When the SCH3 group is replaced by a t-butyl ester group and the resulting analogue is a full agonist in all the above preparations and more active than the parent hexapeptide and SP-OCH3 at NK-1 receptors. It is concluded that for activity at NK-1 receptors methionine can be replaced by gamma-t-butyl glutamate without loss of activity, whilst at NK-2 and NK-3 receptors the above substitution increases the activity of [Orn6]-SP6-11. Other gamma-alkyl esters of the glutamic acid reduce its biological activity.     

Methionine proximity assay,a novel method for exploring peptide ligand-receptor interaction

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms. Most studies of peptidergic GPCRs have been done with photolabeling peptides containing the benzophenone moiety as a reputedly non-selective probe. However our recent results are now showing that p-benzoylphenylalanine (Bpa) has some selectivity for Met residues in the receptor protein, reducing the accuracy of this method. Turning a problem into an asset, modified analogues of Bpa, e.g. p,p'-nitrobenzoylphenylalanine (NO2Bpa), display increased selectivity for such Met residues. It means a photoprobe containing such modified benzophenone-moieties does not label a receptor protein unless a Met residue is in the immediate vicinity. This unique property allows us to propose and show the feasibility and utility of a new method for scanning the contact areas of peptidergic GPCRs, the Methionine Proximity Assay (MPA). Putative contact residues of the receptor are exchanged to Met residues by site-directed mutagenesis and are subjected to photoaffinity labeling with such modified benzophenone-containing peptides. Successful incorporation indicates physical proximity of those residues. This principle is established and explored with benzophenone-containing analogues of angiotensin II and the two known human angiotensin II receptors AT1 and AT2, determining contact points in both receptors. This approach has several important advantages over other scanning approaches, e.g., the SCAM procedure, since the MPA-method can be used in the hydrophobic core of receptors.     

Identification of an interaction between residue 6 of the natural peptide ligand and a distinct residue within the amino-terminal tail of the secretin receptor.

Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding. We recently used this technique to demonstrate the proximity between a residue within the carboxyl-terminal half of a secretin-like ligand and the amino-terminal domain of the secretin receptor (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909). In this work, we have developed another novel radioiodinatable secretin analogue ([Bpa6,Tyr10]rat secretin-27) that incorporates a photolabile p-benzoyl-L-phenylalanine (Bpa) residue into position 6 of the amino-terminal half of the ligand and used this to identify a specific receptor residue proximate to it. This probe specifically bound to the secretin receptor with high affinity (IC50 = 13.2 +/- 2.5 nM) and was a potent stimulant of cAMP accumulation in secretin receptor-bearing Chinese hamster ovary-SecR cells (EC50 = 720 +/- 230 pM). It covalently labeled the secretin receptor in a saturable and specific manner. Cyanogen bromide cleavage of this molecule yielded a single labeled fragment that migrated on an SDS-polyacrylamide gel at Mr = 19,000 that shifted to 10 after deglycosylation, most consistent with either of two glycosylated fragments within the amino-terminal tail. By immunoprecipitation with antibody directed to epitope tags incorporated into each of the two candidate fragments, the most distal fragment at the amino terminus was identified as the domain of labeling. The labeled domain was further refined to the first 16 residues by endoproteinase Lys-C cleavage and by cyanogen bromide cleavage of another receptor construct in which Val16 was mutated to Met. Radiochemical sequencing of photoaffinity-labeled secretin receptor fragments established that Val4 was the specific site of covalent attachment. This provides the first residue-residue contact between a secretin ligand and its receptor and will contribute substantially to the molecular understanding of this interaction.     

Photoaffinity labeling of P450Cam by an imidazole-tethered benzophenone probe

[(3)H]4-Benzoyl-N-[2-(imidazole-4-yl)ethyl]benzamide ([(3)H]HBP) was synthesized and used to photoaffinity label P450(Cam). The imidazole moiety of HBP anchors the compound in the P450(Cam) active site by coordination of the heme iron, thereby insuring that covalent modification occurs in the active site. Additionally, the imidazole anchor provides a known binding orientation of HBP to P450(Cam) from which conclusions about enzyme structure can be drawn based upon the locations of photoadducted residues. Two sites of adduction were identified by MS analysis of digested, photoaffinity labeled P450(Cam). Photoaffinity labeling experiments in the presence of the type II competitive inhibitor, 1-phenylimidazole, were used to assess the specificity of the photoadducts characterized. One adduct was located at Met103 on the flexible B'/C loop region of P450(Cam). The other adduct was localized on the C-helix at Met121. The implications of these data are discussed.     

Direct photoaffinity labeling of leukotriene binding sites

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeling taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock-freezing of the samples, irradiation-induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state. After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time-dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67,000 was labeled. This polypeptide was identified as albumin. Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48,000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48,000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25,000, was identified as subunit 1 of glutathione transferases by immunoprecipitation. The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene-metabolizing enzymes, and transport systems.     

Direct demonstration of unique mode of natural peptide binding to the type 2 cholecystokinin receptor using photoaffinity labeling

Direct analysis of mode of peptide docking using intrinsic photoaffinity labeling has provided detailed insights for the molecular basis of cholecystokinin (CCK) interaction with the type 1 CCK receptor. In the current work, this technique has been applied to the closely related type 2 CCK receptor that also binds the natural full agonist peptide, CCK, with high affinity. A series of photolabile CCK analog probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogs that possessed full biological activity utilized in photoaffinity labeling. The position 29 probe, incorporating a photolabile benzoyl-phenylalanine in that position, was shown to bind with high affinity and to be a full agonist, with potency not different from that of natural CCK, and to covalently label the type 2 CCK receptor in a saturable, specific and efficient manner. Using proteolytic peptide mapping, mutagenesis, and radiochemical Edman degradation sequencing, this probe was shown to establish a covalent bond with type 2 CCK receptor residue Phe¹²⁰ in the first extracellular loop. This was in contrast to its covalent attachment to Glu³⁴⁵ in the third extracellular loop of the type 1 CCK receptor, directly documenting differences in mode of docking this peptide to these receptors.     

Identification of peptide ligand-binding domains within the human motilin receptor using photoaffinity labeling.

The cDNA encoding the human motilin receptor was recently cloned and found to represent a G protein-coupled receptor that is structurally related to the growth hormone secretagogue receptors. Together, these represent a new Class I receptor family. Our aim in the present work is to gain insight into the molecular basis of binding of motilin to its receptor using photoaffinity labeling. To achieve this, we developed a Chinese hamster ovary cell line that overexpressed functional motilin receptor (CHO-MtlR; 175,000 sites per cell, with K(i) = 2.3 +/- 0.4 nm motilin and EC(50) = 0.3 +/- 0.1 nm motilin) and a radioiodinatable peptide analogue of human motilin that incorporated a photolabile p-benzoyl-l-phenylalanine (Bpa) residue into its pharmacophoric domain. This probe, [Bpa(1),Ile(13)]motilin, was a full agonist at the motilin receptor that increased intracellular calcium in a concentration-dependent manner (EC(50) = 1.5 +/- 0.4 nm). This photolabile ligand bound specifically and with high affinity to the motilin receptor (K(i) = 12.4 +/- 1.0 nm), and covalently labeled that molecule within its M(r) = 45,000 deglycosylated core. Cyanogen bromide cleavage demonstrated its covalent attachment to fragments of the receptor having apparent M(r) = 6,000 and M(r) = 31,000. These were demonstrated to represent fragments that included both the first and the large second extracellular loop domains, with the latter representing a unique structural feature of this receptor. The spatial approximation of the pharmacophoric domain of motilin with these receptor domains support their functional importance as well.     

Determination of peptide contact points in the human angiotensin II type I receptor (AT1) with photosensitive analogs of angiotensin II

To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-L-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the [Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166-199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of [Sar1, Bpa8]AngII interacts with the second extracellular loop of the AT1 receptor.