Synthesis and characterization of the first fluorescent nonpeptide NPY Y1 receptor antagonist

Cyanine-5-labelled neuropeptide Y (NPY) was demonstrated to be an ideal universal fluorescent ligand for the combined investigation of NPY Y(1), Y(2) and Y(5) receptors. With respect to improved stability, detection of receptor subtypes in cells and tissues, and prevention of receptor internalization, small nonpeptidic fluorescent antagonists should be superior. Here we present a set of four fluorescent nonpeptide NPY Y(1) receptor (Y(1)R) antagonists. The highest affinity was obtained by labelling an N(G)-(6-aminohexanoyl)argininamide derived from the Y(1)R antagonist BIBP 3226, with Py-1, a small pyrylium dye. The fluorescent pyridinium-type Y(1)R antagonist, compound 4 had K(i) values of 29 nM and 2.7 nM, which were determined by radioligand binding and flow cytometry under equilibrium conditions, respectively; 4 had a K(b) value of 0.6 nM (Ca(2+) assay). The large Stoke's shift (541 vs. 615 nm) in buffer (PBS, pH 7.4) in the presence of 1% BSA and the red emission (quantum yield 56%) are advantageous with respect to the signal-to-noise ratio. The new probe was successfully used in fluorescence-based binding experiments evaluated by flow cytometry and confocal microscopy; this demonstrates the potential of pyrylium dyes for the preparation of fluorescent ligands that are applicable for the study of G protein-coupled receptors on living cells.     

Modeling the G-protein-coupled neuropeptide Y Y1 receptor agonist and antagonist binding sites

Neuropeptide Y (NPY) receptors belong to the G-protein-coupled receptor (GPCR) superfamily and mediate several physiological responses, such as blood pressure, food intake, sedation and memory retention. To understand the interactions between the NPY Y1 receptor subtype and its ligands, computer modeling was applied to the natural peptide agonist, NPY and a small molecule antagonist, BIBP3226. An agonist and antagonist binding domain was elucidated using mutagenesis data for the Y1 receptor as well as for other GPCR families. The agonist and antagonist ligands which were investigated appear to share common residues for their interaction within the transmembrane regions of the Y1 receptor structure, including Gln120, Asn283 and His306. This is in contrast to findings with tachykinin receptors where the binding domains of the non-peptide antagonists have very little in common with the binding domains of the agonist, substance-P. In addition, a hydrogen bond between the hydroxyl group of Tyr36 of NPY and the side chain of Gln219, an interaction that is absent in the model complex between Y1 and the antagonist BIBP3226, is proposed as one of the potential interactions necessary for receptor activation.      

A simple and powerful flow cytometric method for the simultaneous determination of multiple parameters at G protein-coupled receptor subtypes

The quantification of pharmacological parameters at G protein-coupled receptors (GPCRs) is indispensable in drug research but costly and time-consuming when conventional methods are sequentially applied. With neuropeptide Y (NPY) Y(1), Y(2), and Y(5) receptors as model systems, a homogenous flow cytometric method for the simultaneous determination of the affinity, selectivity, and activity of GPCR ligands was developed. Mixtures of cells expressing the receptors of interest and cyanine-labeled NPY as a universal fluorescent Y(1), Y(2), and Y(5) receptor agonist were used. Calcium mobilization was measured in different channels with the aid of fluo-4 and fura red. A combination of dye-loaded HEL-Y(1) and CHO-Y(2)-Galpha(qi5) cells with unloaded HEC-1B-Y(5) cells allowed the simultaneous determination of Y(1), Y(2), and Y(5) receptor selectivity preceded by the Y(1) and Y(2) receptor-mediated response with one and the same sample. The data are in good agreement with those determined by radioligand binding and spectrofluorimetry. The convenient, robust, and inexpensive multiparametric procedure offers a broad range of applications in the pharmacological characterization of GPCR ligands.     

Peptide Architecture: Adding an α‐Helix to the PYY Lysine Side Chain Provides Nanomolar Binding and Body‐Weight‐Lowering Effects

The gut hormone PYY3‐36 influences food intake and body weight via interaction with hypothalamic presynaptic Y2 receptors (Y2R). Novel Y2R‐selective analogues of PYY3‐36 are therefore potential drug candidates for the treatment of obesity. It has been hypothesized that PYY3‐36 and possibly also the related PP‐fold peptides, NPY and PP, bind to the membrane via their amphipathic α‐helix prior to receptor interaction. The PYY3‐36 amphipathic α‐helix causes the peptide to associate with the membrane, making it essential for Y receptor potency as it potentially guides the C‐terminal pentapeptide into the correct conformation for receptor activation. Based on this hypothesis, the importance of the amphipathic nature of PYY3‐36, as well as the ability of amphipathic α‐helices to interact in solution to form di‐ and tetramers, we redesigned the peptide architecture by addition of an amphipathic α‐helix via the Lys 4 side chain of PYY3‐36. Two different amphipathic sequences were introduced; first, PYY17‐31, the native α‐helix of PYY, and secondly, its retro counterpart, PYY31‐17, which is also predicted to form an α‐helix. Moreover, several different turn motifs between the branching point and the additional α‐helix were tested. Several novel peptides with nanomolar Y2R binding affinities, as well as increased Y receptor selectivity, were identified. CD experiments showed the modifications to be well accepted, and an increase in mean ellipticity (ME) signifying an increased degree of α‐helicity was observed. Receptor binding experiments indicated that the direction of the additional α‐helix is less important, in contrast to the turn motifs, which greatly affect the Y1R binding and thus determine the Y1R activity. Conversely, the structure–activity relationships from in vivo data showed that the peptide containing the retro‐sequence was inactive, even though the binding data demonstrated high affinity and selectivity. This demonstrates that radical redesign of peptide architecture can provide nanomolar binding with improved subtype selectivity and with in vivo efficacy.     

Synthethic collagen-like domain derived from the macrophage scavenger receptor binds acetylated low-density lipoprotein in vitro

The bovine macrophage scavenger receptor is a 70 kDa membraneprotein that is trimerized on the macrophage cell surface. Thereceptor binds modified low-density lipoproteins (LDL). Thecore binding site is located within 22 residues at the C-terminusof the collagen-like domain of the receptor. The Lys residueat position 337 plays an important role in ligand binding. Here,the collagen-like domain was constructed using a peptide architecturetechnique, in which three collagenous peptide chains were crosslinkedat their N-termini. The crosslinked peptide showed a collagen-likestructure by circular dichroism and existed mainly in a monomerictriple helical form as shown by gel exclusion chromatography.The triple-stranded peptide was demonstrated to bind acetylatedLDL (Ac-LDL) using regions derived from Gly323 to Lys340 ofthe natural bovine scavenger receptor. However, a single-strandedpeptide with the same amino acid sequence did not bind Ac-LDL.Furthermore, a triple-stranded mutated peptide in which Lyscorresponding to Lys337 in the mother protein was substitutedwith Ala showed no binding activity to Ac-LDL. These results,taken together, indicate that the synthetic collagen-tike peptidehas a similar structure to the binding site in the scavengerreceptor, and support the view that the collagen-tike domainof the natural scavenger receptor recognizes Ac-LDL.     

Position and Length of Fatty Acids Strongly Affect Receptor Selectivity Pattern of Human Pancreatic Polypeptide Analogues

Pancreatic polypeptide (PP) is a satiety‐inducing gut hormone targeting predominantly the Y₄ receptor within the neuropeptide Y multiligand/multireceptor family. Palmitoylated PP‐based ligands have already been reported to exert prolonged satiety‐inducing effects in animal models. Here, we suggest that other lipidation sites and different fatty acid chain lengths may affect receptor selectivity and metabolic stability. Activity tests revealed significantly enhanced potency of long fatty acid conjugates on all four Y receptors with a preference of position 22 over 30 at Y₁, Y₂ and Y₅ receptors. Improved Y receptor selectivity was observed for two short fatty acid analogues. Moreover, [K³⁰(E‐Prop)]hPP_2?36 ( 15 ) displayed enhanced stability in blood plasma and liver homogenates. Thus, short chain lipidation of hPP at key residue 30 is a promising approach for anti‐obesity therapy because of maintained selectivity and a sixfold increased plasma half‐life.     

N-terminal domain of eotaxin-3 is important for activation of CC chemokine receptor 3

Eotaxin-3 belongs to the CC chemokine family, and specificallyrecognizes CC chemokine receptor (CCR) 3 that is expressed oneosinophils, basophils and helper T type 2 cells. The three-dimensionalstructure of eotaxin-3 determined by nuclear magnetic resonancehas revealed that the N-terminal nine residues preceding thefirst cysteine comprise an unstructured domain, which is alsoobserved in other chemokine molecules. In order to determinethe function of the N-terminal domain of eotaxin-3, we constructedvarious N-terminal-deletion mutants, and then examined theirbinding and chemotactic activities toward eosinophils in vitro.Competitive binding studies showed that the binding affinityof truncated mutant toward CCR3 was almost the same as thatof wild-type eotaxin-3 even though the N-terminal truncationinvolved the first through to the ninth residues. In contrast,the chemotactic activity gradually decreased with extensionof the N-terminal deletion, and when the deletion extended tothe eighth residue, the activity was not detected at all. Thus,the N-terminal nine residues are not critical for binding butthe N-terminal eight residues are essential for activation ofCCR3. The truncated eotaxin-3 proteins lacking the N-terminaleight or nine residues inhibited the chemotactic activity ofchemokines that recognize CCR3. The truncated mutants can possiblybe used for anti-allergic and anti-HIV-1 therapy.     

Identification of a Suitable Peptidic Molecular Platform for the Development of NPY(Y1)R-Specific Imaging Agents

NPY(Y₁)R (neuropeptide Y receptor subtype 1) is an important target structure for tumor-specific imaging and therapy as this receptor subtype is overexpressed in very high density and incidence especially in human breast cancer. Targeting this receptor with radiolabeled truncated analogues of the endogenous ligand NPY (neuropeptide Y) has, however, not yet resulted in satisfactory imaging results when using positron emission tomography (PET). This can be attributed to the limited stability of these PET imaging agents caused by their fast proteolytic degradation. Although highly promising NPY analogues were developed, their stability has only been investigated in very few cases. In this systematical work, we comparatively determined the stability of the five most promising truncated analogues of NPY that were developed over the last years, showing the highest receptor affinities and subtype selectivities. The stability of the peptides was assessed in human serum as well as in a human liver microsomal stability assay; these gave complementary results, thus demonstrating the necessity to perform both assays and not just conventional serum stability testing. Of the tested peptides, only [Lys(lauroyl)²⁷,Pro³⁰,Lys(DOTA)³¹,Bip³²,Leu³⁴]NPY_27-36 showed high stability against peptidase degradation; thus this is the best-suited truncated NPY analogue for the development of NPY(Y₁)R-specific imaging agents.     

Molecular modeling of interleukin-8 receptor beta and analysis of the receptor-ligand interaction

A structural model of interleukin-8 receptor type beta (IL-8R-beta) was constructed based on the structure of bacteriorhodopsin. High temperature molecular dynamics simulations were performed to search the possible conformations of loop regions in IL-8R-beta which recognize the ligand. The crystal structure of interleukin 8 (IL-8) was used as a geometric constraint of the extracellular loop regions of IL-8R-beta in the conformational search. 500 complex structures were extracted from the dynamics trajectory and five plausible models were selected based on the binding energy and known experimental data. To study further the interaction between IL-8R-beta and its ligands, the complex of IL-8R- beta and platelet factor 4 (PF4) C-terminal peptide was also modeled by molecular dynamics simulations. From these models, the N-terminus, extracellular domain 3 and extracellular domain 4 of IL-8R-beta were found to be important for ligand binding. Key residues of these regions involved in ligand binding were characterized. These models provide insight into the structural basis of biological activity of IL-8 and PF4 and may guide the design of potential therapeutic agents targeting IL-8 receptors. Furthermore, the approach developed from this study may have implications for the understanding of other chemokine receptor- ligand interactions that have been recently suggested to be involved in HIV infection.      

Semisynthesis and characterization of the first analogues of pro-neuropeptide y

Enzymatic cleavage of prohormone neuropeptide Y (proNPY) leads to mature neuropeptide Y (NPY), a widely distributed neuropeptide with multiple functions both peripherally and centrally. A single dibasic pair of amino acids, Lys38-Arg39, represents the recognition motif for a class of hormone-processing enzymes known as prohormone convertases (PCs). Two members of this PC family, PC1/3 and PC2, are involved in proNPY cleavage. The aim of this work was to establish an effective method for the generation of full-length 69-amino acid proNPY analogues for further studies of prohormone convertase interaction. We have chosen two ligation sites in order to perform the semisynthesis of proNPY analogues by expressed protein ligation (EPL). By using the intein-mediated purification system (IMPACT) with improved conditions for intein splicing, we were able to isolate proNPY 1-40 and proNPY 1-54 fragments as Cterminal thioesters. Peptides bearing Nterminal cysteine instead of the naturally occurring Ser41 and Thr55 residues, respectively, were generated by solid-phase peptide synthesis. Moreover, labels (carboxyfluorescein and biotin) were inserted into the peptide sequences. The synthesis of the [C41]proNPY 41-69 fragment, which proved to be a difficult peptide sequence, could be achieved by the incorporation of two pseudo-proline derivatives. Western blot analysis revealed that all five proNPY analogues are recognized by monoclonal antibodies directed against NPY as well as against the Cflanking peptide of NPY (CPON).     

Molecular modelling of the ORL1 receptor and its complex with nociceptin

The opioid receptor like (ORL1) receptor is a G-protein coupled receptor superfamily, and regulates a plethora of neurophysiological functions. The structural requirements for receptor activation by its endogenous agonist, nociceptin (FGGFTGARKSARKLANQ), differ markedly from those of the kappa-opioid receptor and its putative peptide agonist, dynorphin A (YGGFLRRIRPKLKWDNQ). In order to probe the functional architecture of the ORL1 receptor, a molecular model of the receptor has been built, including the TM domain and the extra- and intracellular loops. An extended binding site able to accommodate nociceptin-(1-13), the shortest fully active analogue of nociceptin, has been characterized. The N-terminal FGGF tetrapeptide is proposed to bind in a highly conserved region, comprising two distinct hydrophobic pockets in a cavity formed by TM helices 3, 5, 6 and 7, capped by the acidic second extracellular (EL2) loop controlling access to the TM elements of the peptide binding site. The nociceptin conformation provides for the selective preference of the ORL1 receptor for nociceptin over dynorphin A, conferred by residue positions 5 and 6 (TG versus LR), and the favourable interaction of its highly positively charged core (residues 8-13) with the EL2 loop, thought to mediate receptor activation. The functional roles of the EL2 loop and the conserved N-terminal tetrapeptide opioid 'message' binding site are discussed in the context of the different structural requirements of the ORL1 and kappa-opioid receptors for activation.      

Conformationally constrained CCK8 analogues obtained from a rationally designed peptide library as ligands for cholecystokinin type B receptor

A library of 14 cyclic peptide analogues derived from the octapeptide C-terminal sequence of the human cholecystokinin hormone (CCK(26-33), or CCK8) was designed, synthesized, and characterized. The 14 peptide analogues were rationally designed to specifically interact with the CCK type B receptor (CCK(B)-R) on the basis of the structure of the bimolecular complex between CCK8 and the third extracellular loop of CCK(B)-R, namely CCK(B)-R(352-379). The rational design of new ligands for CCK(B)-R has relied on stabilization by cyclic constraints of the structural motifs that bring the key residues of the ligand (especially Trp 30, Met 31, and Phe 33) in the proper spatial orientation for optimal interaction with the receptor. The binding affinity of the new ligands for CCK(B)-R was assessed by displacement experiments of (111)In-radiolabeled CCK8 in cells that overexpress the CCK(B) receptor. The new ligands generally showed binding affinities lower than that of parent CCK8, with the best compounds having IC50 values around 10 microM. Structure-activity relationship data show that preservation of the Trp 30-Met 31 motif is essential and that the Phe 33 side chain must be present. NMR conformational studies of the compound with maximal binding affinity (cyclo-B11, IC50=11 microM) in DPC micelles shows that this compound presents a turn-like conformation centered at the Trp 30-Met 31 segment, as planned by rational design. Such a conformation is stabilized by its interaction with the micelle rather than by the cyclic constraint.     

Biosynthesis and spectroscopic characterization of 2-TM fragments encompassing the sequence of a human GPCR, the Y4 receptor

This paper presents a divide‐and‐conquer approach towards obtaining solution structures of G protein‐coupled receptors. The human Y4 receptor was dissected into two to three transmembrane helix fragments, which were individually studied by solution NMR. We systematically compared various biosynthetic routes for the expression of the fragments in Escherichia coli and discuss purification strategies. In particular, we have compared the production of transmembrane (TM) fragments as inclusion bodies by using the ΔTrp leader sequence, with membrane‐directed expression by using Mistic as the fusion partner, and developed methods for enzymatic cleavage. In addition, direct expression of two‐TM fragments into inclusion bodies is a successful route in some cases. With the exception of TM13, we could produce all fragments in isotope‐labeled form in quantities sufficient for NMR studies. Almost complete backbone resonance assignment was obtained for the first two helices, as well as for helices 5 and 7, and a high degree was obtained for TM6, while conformational exchange processes resulted in the disappearance of many signals from TM4. In addition, complete assignments were obtained for all residues of the N‐terminal domain, as well as the extracellular and cytosolic loops (with the exception of an undecapeptide segment in the second extracellular loop, EC2) and for the complete cytosolic C‐terminal tail. In total, backbone resonances of 78 % of all residues were assigned for the Y4 receptor. Predictions of secondary structure based on backbone chemical shifts indicate that most residues from the TM regions adopt helical conformations, with exception of those around polar residues or prolines. However, the domain boundaries differ slightly from those predicted for homology models. We suggest that the obtained chemical shifts might be useful in assigning the full‐length receptor.     

Unexpected opioid activity profiles of analogues of the novel peptide kappa opioid receptor ligand CJ-15,208

An alanine scan was performed on the novel κ opioid receptor (KOR) peptide ligand CJ‐15,208 to determine which residues contribute to the potent in vivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combination of solid‐phase peptide synthesis of the linear precursors, followed by cyclization in solution. Like the parent peptide, each of the analogues exhibited agonist activity and KOR antagonist activity in an antinociceptive assay in vivo. Unlike the parent peptide, the agonist activity of the potent analogues was mediated predominantly, if not exclusively, by μ opioid receptors (MOR). Thus analogues 2 and 4 , in which one of the phenylalanine residues was replaced by alanine, exhibited both potent MOR agonist activity and KOR antagonist activity in vivo. These peptides represent novel lead compounds for the development of peptide‐based opioid analgesics.     

Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity

Human galanin is a 30 amino acid neuropeptide that elicits arange of biological activities by interaction with G protein-coupledreceptors. We have generated a model of the human GALR1 galaninreceptor subtype (hGALR1) based on the alpha carbon maps offrog rhodopsin and investigated the significance of potentialcontact residues suggested by the model using site-directedmutagenesis. Mutation of Phe186 within the second extracellularloop to Ala resulted in a 6-fold decrease in affinity for galanin,representing a change in free energy consistent with hydrophobicinteraction. Our model suggests interaction between Phe186 ofhGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificitywas investigated by replacement of residues in hGALR1 with thecorresponding residues in hGALR2 and use of the hGALR2-specificligands hGalanin(2–30) and [D-Trp2]hGalanin(1–30).The His267Ile mutant receptor exhibited a pharmacological profilecorresponding to that of hGALR1, suggesting that His267 is notinvolved in a receptor–ligand interaction. The mutationPhe115Ala resulted in a decreased binding affinity for hGalaninand for hGALR2-specific analogues, indicating Phe115 to be ofstructural importance to the ligand binding pocket of hGALR1but not involved in direct ligand interaction. Analysis of Glu271Trpsuggested that Glu271 of hGALR1 interacts with the N-terminusof galanin and that the Trp residue in the corresponding positionin hGALR2 is involved in receptor subtype specificity of binding.Our model supports previous reports of Phe282 of hGALR1 interactingwith Trp2 of galanin and His264 of hGALR1 interacting with Tyr9of galanin.     

A cyclic CCK8 analogue selective for the cholecystokinin type A receptor: design, synthesis, NMR structure and binding measurements

A cyclic CCK8 analogue, cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 (Dpr=L-2,3-diaminopropionic acid), has been designed on the basis of the NMR structure of the bimolecular complex between the N-terminal fragment of the CCK(A) receptor and its natural ligand CCK8. The conformational features of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 have been determined by NMR spectroscopy in aqueous solution and in water containing DPC-d(38) micelles (DPC=dodecylphosphocholine). The structure of the cyclic peptide in aqueous solution is found to be in a relaxed conformation, with the backbone and Dpr29 side chain atoms making a planar ring and the N-terminal tripeptide extending approximately along the plane of this ring. In DPC/water, the cyclic peptide adopts a "boat-shaped" conformation, which is more compact than that found in aqueous solution. The cyclic constraint between the Dpr29 side chain and the CCK8 carboxyl terminus (Lys34) introduces a restriction in the backbone conformational freedom. However, the interaction of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 with the micelles still plays an important role in the stabilisation of the bioactive conformation. A careful comparison of the NMR structure of the cyclic peptide in a DPC micelle aqueous solution with the structure of the rationally designed model underlines that the turn-like conformation in the Trp30-Met31 region is preserved, such that the Trp30 and Met31 side chains can adopt the proper spatial orientation to interact with the CCK(A) receptor. The binding properties of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 to the N-terminal receptor fragment have been investigated by fluorescence spectroscopy in a micellar environment. Estimates of the apparent dissociation constant, K(d), were in the range of 70-150 nM, with a mean value of 120+/-27 nM. Preliminary nuclear medicine studies on cell lines transfected with the CCK(A) receptor indicate that the sulfated-Tyr derivative of cyclo(29,34)[Dpr(29),Lys(34)]-CCK8 displaces the natural ligand with an IC(50) value of 15 microM.     

The predicted 3D structures of the human M1 muscarinic acetylcholine receptor with agonist or antagonist bound

The muscarinic acetylcholine G-protein-coupled receptors are implicated in diseases ranging from cognitive dysfunctions to smooth-muscle disorders. To provide a structural basis for drug design, we used the MembStruk computational method to predict the 3D structure of the human M1 muscarinic receptor. We validated this structure by using the HierDock method to predict the binding sites for three agonists and four antagonists. The intermolecular ligand-receptor contacts at the predicted binding sites agree well with deductions from available mutagenesis experiments, and the calculated relative binding energies correlate with measured binding affinities. The predicted binding site of all four antagonists is located between transmembrane (TM) helices 3, 4, 5, 6, and 7, whereas the three agonists prefer a site involving residues from TM3, TM6, and TM7. We find that Trp 157(4) contributes directly to antagonist binding, whereas Pro 159(4) provides an indirect conformational switch to position Trp 157(4) in the binding site (the number in parentheses indicates the TM helix). This explains the large decrease in ligand binding affinity and signaling efficacy by mutations of Trp 157(4) and Pro 159(4) not previously explained by homology models. We also found that Asp 105(3) and aromatic residues Tyr 381(6), Tyr 404(7), and Tyr 408(7) are critical for binding the quaternary ammonium head group of the ligand through cation-pi interactions. For ligands with a charged tertiary amine head group, we suggest that proton transfer from the ligand to Asp 105(3) occurs upon binding. Furthermore, we found that an extensive aromatic network involving Tyr 106(3), Trp 157(4), Phe 197(5), Trp 378(6), and Tyr 381(6) is important in stabilizing antagonist binding. For antagonists with two terminal phenyl rings, this aromatic network extends to Trp 164(4), Tyr 179(extracellular loop 2), and Phe 390(6) located at the extracellular end of the TMs. We find that Asn 382(6) forms hydrogen bonds with selected antagonists. Tyr381(6) and Ser 109(3) form hydrogen bonds with the ester moiety of acetylcholine, which binds in the gauche conformation.     

Receptor‐Mediated Uptake of Boron‐Rich Neuropeptide Y Analogues for Boron Neutron Capture Therapy

Peptidic ligands selectively targeting distinct G protein‐coupled receptors that are highly expressed in tumor tissue represent a promising approach in drug delivery. Receptor‐preferring analogues of neuropeptide Y (NPY) bind and activate the human Y₁ receptor subtype (hY₁ receptor), which is found in 90 % of breast cancer tissue and in all breast‐cancer‐derived metastases. Herein, novel highly boron‐loaded Y₁‐receptor‐preferring peptide analogues are described as smart shuttle systems for carbaboranes as ¹⁰B‐containing moieties. Various positions in the peptide were screened for their susceptibility to carbaborane modification, and the most promising positions were chosen to create a multi‐carbaborane peptide containing 30 boron atoms per peptide with excellent activation and internalization patterns at the hY₁ receptor. Boron uptake studies by inductively coupled plasma mass spectrometry revealed successful uptake of the multi‐carbaborane peptide into hY₁‐receptor‐expressing cells, exceeding the required amount of 10⁹ boron atoms per cell. This result demonstrates that the NPY/hY receptor system can act as an effective transport system for boron‐containing moieties.     

Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units

The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs) are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY) receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY) receptors and could apply to many receptors that use large peptidic agonists.