Birgitte Holst

Common structural basis for constitutive activity of the ghrelin receptor family

Three members of the ghrelin receptor family were characterized in parallel: the ghrelin receptor, the neurotensin receptor 2 and the orphan receptor GPR39. In transiently transfected COS-7 and human embryonic kidney 293 cells, all three receptors displayed a high degree of ligand-independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the Gq, phospholipase C pathway was ∼50% of their maximal capacity as determined through inositol phosphate accumulation. These two receptors also showed very high constitutive activity in activation of cAMP response element-driven transcription. GPR39 displayed a clear but lower degree of constitutive activity through the inositol phosphate and cAMP response element pathways. In contrast, GPR39 signaled with the highest constitutive activity in respect of activation of serum response element-dependent transcription, in part, possibly, through G12/13 and Rho kinase. Antibody feeding experiments demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically be tuned up and down depending on the size and hydrophobicity of the side chain in position VI:16 in the context of an aromatic residue at VII:09 and a large hydrophobic residue at VII:06. It is concluded that the three ghrelin-like receptors display an unusually high degree of constitutive activity, the structural basis for which is determined by an aromatic cluster on the inner face of the extracellular ends of TMs VI and VII.

Ghrelin receptor mutations - : too little height and too much hunger

The ghrelin receptor is known from in vitro studies to signal in the absence of the hormone ghrelin at almost 50% of its maximal capacity. But, as for many other 7-transmembrane receptors, the in vivo importance of this ligand-independent signaling has remained unclear. In this issue of the JCI, Pantel et al. find that a natural mutation in the ghrelin receptor, Ala204Glu, which is associated with a selective loss of constitutive activity without affecting ghrelin affinity, potency, or efficacy, segregates in 2 families with the development of short stature (see the related article beginning on page 760). By combination of the observations from this study with those related to the phenotype of subjects carrying another natural ghrelin receptor mutation, Phe279Leu, having identical molecular-pharmacological properties, it is proposed that selective lack of ghrelin receptor constitutive signaling leads to a syndrome characterized not only by short stature, but also by obesity that apparently develops during puberty.

Neurotensin Is Coexpressed, Coreleased, and Acts Together With GLP-1 and PYY in Enteroendocrine Control of Metabolism.

The 2 gut hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are well known to be coexpressed, costored, and released together to coact in the control of key metabolic target organs. However, recently, it became clear that several other gut hormones can be coexpressed in the intestinal-specific lineage of enteroendocrine cells. Here, we focus on the anatomical and functional consequences of the coexpression of neurotensin with GLP-1 and PYY in the distal small intestine. Fluorescence-activated cell sorting analysis, laser capture, and triple staining demonstrated that GLP-1 cells in the crypts become increasingly multihormonal, ie, coexpressing PYY and neurotensin as they move up the villus. Proglucagon promoter and pertussis toxin receptor-driven cell ablation and reappearance studies indicated that although all the cells die, the GLP-1 cells reappear more quickly than PYY- and neurotensin-positive cells. High-resolution confocal fluorescence microscopy demonstrated that neurotensin is stored in secretory granules distinct from GLP-1 and PYY storing granules. Nevertheless, the 3 peptides were cosecreted from both perfused small intestines and colonic crypt cultures in response to a series of metabolite, neuropeptide, and hormonal stimuli. Importantly, neurotensin acts synergistically, ie, more than additively together with GLP-1 and PYY to decrease palatable food intake and inhibit gastric emptying, but affects glucose homeostasis in a more complex manner. Thus, neurotensin is a major gut hormone deeply integrated with GLP-1 and PYY, which should be taken into account when exploiting the enteroendocrine regulation of metabolism pharmacologically.

Molecular mechanism of Zn2+ agonism in the extracellular domain of GPR39

Ala substitution of potential metal‐ion binding residues in the main ligand‐binding pocket of the Zn2+‐activated G protein‐coupled receptor 39 (GPR39) receptor did not decrease Zn2+ potency. In contrast, Zn2+ stimulation was eliminated by combined substitution of His17 and His19, located in the N‐terminal segment. Surprisingly, substitution of Asp313 located in extracellular loop 3 greatly increased ligand‐independent signaling and apparently eliminated Zn2+‐induced activation. It is proposed that Zn2+ acts as an agonist for GPR39, not in the classical manner by directly stabilizing an active conformation of the transmembrane domain, but instead by binding to His17 and His19 in the extracellular domain and potentially by diverting Asp313 from functioning as a tethered inverse agonist through engaging this residue in a tridentate metal‐ion binding site.

Natural agonist enhancing bis-His zinc-site in transmembrane segment V of the tachykinin NK3 receptor

In the wild‐type tachykinin NK3A receptor histidyl residues are present at two positions in TM‐V, V:01 and V:05, at which Zn2+ functions as an antagonist in NK1 and κ‐opioid receptors with engineered metal‐ion sites. Surprisingly, in the NK3A receptor Zn2+ instead increased the binding of the agonist 125I‐[MePhe7]neurokinin B to 150%. [MePhe7]neurokinin B bound to the NK3A receptor in a two‐component mode of which Zn2+ eliminated the subnanomolar binding mode but induced a higher binding capacity of the nanomolar binding mode. Signal transduction was not induced by ZnCl2 but 10 μM ZnCl2 enhanced the effect of neurokinin B. Ala‐substitution of HisV:01 eliminated the enhancing effect of Zn2+ on peptide binding. It is concluded that physiological concentrations of Zn2+ have a positive modulatory effect on the binding and function of neurokinin B on the NK3A receptor through a bis‐His site in TM‐V.

PheVI:09 (Phe6.44) as a Sliding Microswitch in Seven-transmembrane (7TM) G Protein-coupled Receptor Activation

Background: PheVI:09 (6.44) is highly conserved among 7TM receptors. Results: In the inactive state, PheVI:09 is locked against the backbone of TM-III but slides pass IleIII:16 (3.40) into a tight hydrophobic pocket between TM-III and TM-V during receptor activation. Conclusion: Mutational analysis and computational chemistry shows that PheVI:09 functions as a microswitch. Significance: This work clarifies the molecular mechanism of a crucial microswitch in 7TM receptor activation. In seven-transmembrane (7TM), G protein-coupled receptors, highly conserved residues function as microswitches, which alternate between different conformations and interaction partners in an extended allosteric interface between the transmembrane segments performing the large scale conformational changes upon receptor activation. Computational analysis using x-ray structures of the β2-adrenergic receptor demonstrated that PheVI:09 (6.44), which in the inactive state is locked between the backbone and two hydrophobic residues in transmembrane (TM)-III, upon activation slides ∼2 Å toward TM-V into a tight pocket generated by five hydrophobic residues protruding from TM-III and TM-V. Of these, the residue in position III:16 (3.40) (often an Ile or Val) appears to function as a barrier or gate for the transition between inactive and active conformation. Mutational analysis showed that PheVI:09 is essential for the constitutive and/or agonist-induced signaling of the ghrelin receptor, GPR119, the β2-adrenergic receptor, and the neurokinin-1 receptor. Substitution of the residues constituting the hydrophobic pocket between TM-III and TM-V in the ghrelin receptor in four of five positions impaired receptor signaling. In GPR39, representing the 12% of 7TM receptors lacking an aromatic residue at position VI:09, unchanged agonist-induced signaling was observed upon Ala substitution of LeuVI:09 despite reduced cell surface expression of the mutant receptor. It is concluded that PheVI:09 constitutes an aromatic microswitch that stabilizes the active, outward tilted conformation of TM-VI relative to TM-III by sliding into a tight hydrophobic pocket between TM-III and TM-V and that the hydrophobic residue in position III:16 constitutes a gate for this transition.

Modulation of constitutive activity and signaling bias of the ghrelin receptor by conformational constraint in the second extracellular loop

Background: A natural Glu for Ala variant in the ghrelin receptor extracellular loop 2 selectively eliminates constitutive signaling. Results: Computational chemistry and mutational analysis show that charged residues and metal ion sites that induce α-helix formation in ECL2 prevent constitutive signaling. Conclusion: Flexibility of ECL2 connecting TM-III and TM-V is essential for spontaneous receptor signaling. Significance: Clarification of ECL2 structural constraint is important for receptor signaling. Based on a rare, natural Glu for Ala-204(C+6) variant located six residues after the conserved Cys residue in extracellular loop 2b (ECL2b) associated with selective elimination of the high constitutive signaling of the ghrelin receptor, this loop was subjected to a detailed structure functional analysis. Introduction of Glu in different positions demonstrated that although the constitutive signaling was partly reduced when introduced in position 205(C+7) it was only totally eliminated in position 204(C+6). No charge-charge interaction partner could be identified for the Glu(C+6) variant despite mutational analysis of a number of potential partners in the extracellular loops and outer parts of the transmembrane segments. Systematic probing of position 204(C+6) with amino acid residues of different physicochemical properties indicated that a positively charged Lys surprisingly provided phenotypes similar to those of the negatively charged Glu residue. Computational chemistry analysis indicated that the propensity for the C-terminal segment of extracellular loop 2b to form an extended α-helix was increased from 15% in the wild type to 89 and 82% by introduction in position 204(C+6) of a Glu or a Lys residue, respectively. Moreover, the constitutive activity of the receptor was inhibited by Zn2+ binding in an engineered metal ion site, stabilizing an α-helical conformation of this loop segment. It is concluded that the high constitutive activity of the ghrelin receptor is dependent upon flexibility in the C-terminal segment of extracellular loop 2 and that mutations or ligand binding that constrains this segment and thereby conceivably the movements of transmembrane domain V relative to transmembrane domain III inhibits the high constitutive signaling.

Modifying the conserved C‐terminal tyrosine of the peptide hormone PYY3‐36 to improve Y2 receptor selectivity

The Y2 selective PYY derived peptide PYY3‐36 was recently shown to play a role in appetite regulation. Novel PYY3‐36 analogs with high selectivity for the Y2 receptor could be potential drug candidates for the treatment of obesity. The C‐terminal pentapeptide segment of PYY3‐36 is believed to bind to the Y receptors. Tyr‐36 is highly conserved across species and only few successful modifications of Tyr‐36 have been documented. PYY3‐36 analogs were prepared using solid‐phase peptide chemistry and tested for binding to the Y1, Y2 and Y4 receptor subtypes by radioligand displacement assay. The Y2 receptor agonists with the best affinity and selectivity were further investigated for activity towards the Y1 and Y2 receptor subtypes. Unexpectedly, modifications of Tyr‐36 were well‐tolerated, and the analogs of PYY3‐36 in which the Tyr‐36 hydroxyl group was substituted with a halogen or an amino group were particularly well tolerated and yielded an improved selectivity and approximately equipotent affinity to the Y2 receptor. These modifications could be used to design new potential drug candidates for the treatment of obesity. Copyright

Improving membrane binding as a design strategy for amphipathic peptide hormones: 2‐helix variants of PYY3‐36

It has been hypothesized that amphipathic peptides might bind to membranes prior to activating their cognate receptors, but this has proven difficult to test. The peptide hormone PYY3‐36 is believed to perform its appetite‐suppressing actions through binding to hypothalamic Y2 receptors. It has been proposed that PYY3‐36 via its amphipathic α‐helix binds to the plasma membrane prior to receptor docking. Here, our aim was to study the implication of this hypothesis using new analogs of PYY3‐36. We first studied membrane binding of PYY3‐36. Next, we designed a series of PYY3‐36 analogs to increase membrane‐binding affinity by substituting the N‐terminal segment with a de novo designed α‐helical, amphipathic sequence. These 2‐helix variants of PYY3‐36 were assembled by solid‐phase peptide synthesis. Pharmacological studies demonstrated that even though the native peptide sequence was radically changed, highly active Y2 receptor agonists were generated. A potent analog, with a Kd of 4u2009nM for membranes, was structurally characterized by NMR in the membrane‐bound state, which clearly showed that it formed the expected 2‐helix. The topology of the peptide–micelle association was studied by paramagnetic relaxation enhancement using a spin label, which confirmed that the hydrophobic residues bound to the membrane. Our studies further support the hypothesis that PYY3‐36 associates with the membrane and indicate that this can be used in the design of novel molecules with high receptor binding potency. These observations are likely to be generally important for peptide hormones and biopharmaceutical drugs derived from them. This new 2‐helix variant of PYY3‐36 will be useful as a tool compound for studying peptide–membrane interactions. Copyright