Demonstration of a Specific Site of Covalent Labeling of the Human Motilin Receptor Using a Biologically Active Photolabile Motilin Analog

Abstract

The motilin receptor belongs to a group of class I G proteincoupled receptors that also includes the growth hormone secretagogue and ghrelin receptors. These represent clinically useful targets for pharmacotherapy. Their potentially unique structures and the molecular basis of their binding are not yet clear. We previously reported the initial affinity labeling of a region within this receptor (a cyanogen bromide fragment extending from the first to the second extracellular loop) using a position 1 photolabile motilin analog. To extend our understanding of the molecular basis of motilin binding, we have developed an additional radioiodinatable motilin analog probe having site of covalent attachment in position 5. This was a full agonist that bound to the motilin receptor specifically and with high affinity, and that efficiently established a single covalent bond to its receptor. Sequential chemical and enzymatic cleavage of labeled wild-type and mutant motilin receptor constructs established that the region of labeling was within the third extracellular loop. This was further localized to Phe 332 using radiochemical Edman degradation sequencing. These data provide the first spatial approximation constraint that can be used in the docking of this peptide ligand to its receptor. We hope that a series of such constraints can be determined to provide adequate structural information to begin to elucidate the conformation of this agonist-bound receptor and to ultimately be useful in the rational design of drugs acting at this important target.The detailed understanding of the basis for binding a ligand by a receptor can help refine our understanding of the conformation of the receptor and of the molecular basis for its activation. Although all members of the superfamily of guanine nucleotide-binding protein (G protein)-coupled receptors have structural similarities, with seven hydrophobic segments that are believed to traverse the plasma membrane, the details of even this most fundamental characteristic varies within the family of these receptors (Kolakowski, 1994;Ji et al., 1998). This superfamily is also remarkable for the structural diversity of the natural ligands that activate various members, with themes ranging from small photons and biogenic amines that bind within the confluence of transmembrane segments to larger peptide and glycoprotein ligands that bind to extracellular tail and loop domains.The class I G protein-coupled receptors that are activated by motilin and ghrelin were recognized only recently (Howard et al., 1996;McKee et al., 1997;Feighner et al., 1999) and cluster together in sequence analysis, having substantial structural similarity. This has typically been a predictor of similarity in ligand structure and mechanism of binding and activation (Kolakowski, 1994). Although these natural ligands have extensive sequence homology, ghrelin requires a unique post-translational modification for its activity that is not present or required for motilin (Kojima et al., 1999). This represents the N...

Differential Contributions of Motilin Receptor Extracellular Domains for Peptide and Non-peptidyl Agonist Binding and Activity

“…These results are nicely supported by the results of photoaffinity labeling studies in which ligand probes were used that had photolabile benzoylphenylalanine sites of covalent attachment in positions one and five (8,9). The former probe labeled motilin receptor regions between residues 99 and 129, including the first extracellular loop domain, and between residues 130 and 185, including the beginning of the second extracellular loop (8).…”

“…4D). This is consistent with our most recent identification of this residue as the site of covalent labeling with a photolabile motilin analogue incorporating a benzoylphenylalanine in position 5 (9).…”

“…The broad and diffuse base for natural motilin action is also further defined and found to be consistent with regions identified in photoaffinity labeling studies using probes with photolabile sites of covalent attachment in motilin peptide residues 1 and 5 (8,9). This work also adds insights into key structural features of this receptor, confirming the presence of a disulfide bond that is highly conserved across this superfamily (10,11) and drawing attention to another likely bond in the amino-terminal tail linking Cys 25 and Cys…”

“…The former probe labeled motilin receptor regions between residues 99 and 129, including the first extracellular loop domain, and between residues 130 and 185, including the beginning of the second extracellular loop (8). The latter probe was shown to specifically label Phe 332 in the third extracellular loop domain (9). The other interesting and important insight coming from the current work is the clear establishment of distinct mechanisms of binding and action of peptide and non-peptidyl agonists acting at the motilin receptor.…”

“…Current insights into the mechanism of binding of motilin have come from receptor mutagenesis and photoaffinity labeling studies (7)(8)(9). The only existing mutagenesis studies have carefully examined the predicted second extracellular loop region of the motilin receptor (7).…”