Localization of a Single Transglutaminase-Reactive Glutamine in the Third Domain of RAP, the α2-Macroglobulin Receptor-Associated Protein

The 39-kDa receptor-associated protein (RAP) is an intracellular glycoprotein that interacts with hitherto unknown sites in several members of the low-density-lipoprotein receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. In the present study, the transglutaminase-catalyzed incorporation of radioactively labeled putrescine and a dansylated glutamine-containing peptide into human RAP has been studied. The results indicate the presence of both glutamine and lysine residues in RAP, accessible for transglutaminase cross-linking. Moreover, enzymatic digestion followed by sequence analysis of radiolabeled fractions demonstrated that Gln²⁶¹ acts as the amine acceptor site. This residue is located in the third domain of RAP and is conserved among the RAP interspecies homologues. Insertion of a reporter group into the protein could prove useful to assess ligand/receptor interactions.     

The carboxyl-terminal domain of receptor-associated protein facilitates proper folding and trafficking of the very low density lipoprotein receptor by interaction with the three amino-terminal ligand-binding repeats of the receptor.

The 39-kDa receptor-associated protein (RAP) is a specialized antagonist that inhibits all known ligand interactions with receptors that belong to the low density lipoprotein (LDL) receptor gene family. Recent studies have demonstrated a role for RAP as a molecular chaperone for the LDL receptor-related protein during receptor folding and trafficking within the early secretory pathway. In the present study, we investigated a potential role for RAP as a chaperone for the very low density lipoprotein (VLDL) receptor, another member of the LDL receptor gene family. Using intracellular cross-linking techniques, we found that RAP is associated with newly synthesized VLDL receptor. In the absence of RAP co-expression, newly synthesized VLDL receptor exhibited slower trafficking along the early secretory pathway, most likely due to misfolding of the receptor. The role of RAP in the folding of the VLDL receptor was further studied using an anchor-free, soluble VLDL receptor. Metabolic pulse-chase labeling experiments showed that while only 3% of the soluble VLDL receptor was folded and secreted in the absence of RAP co-expression, over 50% of the soluble receptor was secreted in the presence of RAP co-expression. The functions of RAP in VLDL receptor folding and trafficking were mediated by its carboxyl-terminal repeat but not by the amino-terminal and central repeats. Using truncated VLDL receptor constructs, we identified the RAP-binding site within the first three ligand-binding repeats of the VLDL receptor. Thus, our present study demonstrates that RAP serves as a folding and trafficking chaperone for the VLDL receptor via interactions of its carboxyl-terminal repeat with the three amino-terminal ligand-binding repeats of the VLDL receptor.     

39 kDa receptor-associated protein is an ER resident protein and molecular chaperone for LDL receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic receptor with the ability to bind and endocytose several structurally and functionally distinct ligands. A 39 kDa receptor-associated protein (RAP) inhibits all ligand interactions with LRP in vitro. In the present study, we demonstrate that RAP is an endoplasmic reticulum (ER) resident protein. The tetrapepetide sequence HNEL at the C-terminus of RAP is both necessary and sufficient for RAP retention within the ER. Metabolic labeling combined with cross-linking studies show that RAP interacts with LRP in vivo. Pulse-chase analysis reveals that this association is transient early in the secretory pathway and coincides with LRP aggregation and reduced ligand binding activity. Both internal triplicated LRP binding domains on RAP and multiple RAP binding domains on LRP appear to contribute to the aggregation of LRP and RAP. Dissociation of RAP from LRP results from the lower pH encountered later in the secretory pathway and correlates with an increase in LRP ligand binding activity. Taken together, our results thus suggest that RAP functions intracellularly as a molecular chaperone for LRP and regulates its ligand binding activity along the secretory pathway.     

RAP,a specialized chaperone,prevents ligand-induced ER retention and degradation of LDL receptor-related endocytic receptors.

The multifunctional low density lipoprotein (LDL) receptor-related protein (LRP) forms a complex with a receptor-associated protein (RAP) within the secretory pathway. RAP inhibits ligand binding to LRP and is required for normal functional expression of LRP in vivo, suggesting a physiological function as a specialized chaperone. We have used RAP-deficient mice, generated by gene targeting, to investigate the role of RAP in the biosynthesis and biological activity of LRP and other members of the LDL receptor gene family in various organs and in embryonic fibroblasts. Our results demonstrate that RAP is required for the proper folding and export of the receptors from the endoplasmic reticulum (ER) by preventing the premature binding of co-expressed ligands. Overexpression of apolipoprotein E (apoE), a high affinity ligand for LRP, results in dramatically reduced cellular LRP expression, an effect that is prevented by co-expression of RAP. RAP thus defines a novel class of molecular chaperones that selectively protect endocytic receptors by binding to newly synthesized receptor polypeptides, thereby preventing ligand-induced aggregation and subsequent degradation in the ER.     

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Low density lipoprotein receptor (LDLR) was shown to mediate clearance of blood coagulation factor VIII (FVIII) from the circulation. To elucidate the mechanism of interaction of LDLR and FVIII, our objective was to identify the region of the receptor necessary for binding FVIII. Using surface plasmon resonance, we found that LDLR exodomain and its cluster of complement-type repeats (CRs) bind FVIII in the same mode. This indicated that the LDLR site for FVIII is located within the LDLR cluster. Similar results were obtained for another ligand of LDLR, α-2-macroglobulin receptor-associated protein (RAP), a common ligand of receptors from the LDLR family. We further generated a set of recombinant fragments of the LDLR cluster and assessed their structural integrity by binding to RAP and by circular dichroism. A number of fragments overlapping CR.2-5 of the cluster were positive for binding RAP and FVIII. The specificity of these interactions was tested by site-directed mutagenesis of conserved tryptophans within the LDLR fragments. For FVIII, the specificity was also tested using a single-chain variable antibody fragment directed against the FVIII light chain as a competitor. Both cases resulted in decreased binding, thus confirming its specificity. The mutagenic study also showed an importance of the conserved tryptophans in LDLR for both ligands, and the competitive binding results showed an involvement of the light chain of FVIII in its interaction with LDLR. In conclusion, the region of CR.2-5 of LDLR was defined as the binding site for FVIII and RAP.     

Functional mimicry of the LDL receptor-associated protein through multimerization of a minimized third domain

The LDL receptor-associated protein (RAP) is a ligand for the LDL receptor-related protein (LRP1). The first and third domains of RAP can each bind to one of many sequence-related pairs of complement-type repeats (CR) found within the LRP1 ectodomain. Multiple sites of interaction between the multivalent RAP ligand and the multivalent LRP1 receptor yield strong binding avidity for the complex. The third domain of RAP can be significantly truncated, with material retention of monovalent CR pair-binding affinity, provided that the minimized sequence is stabilized with an intramolecular disulfide bond. We demonstrate that the avidity of full-length RAP for LRP1 in vitro can be partially reconstituted by assembly of truncated, disulfide-linked RAP peptides on tetravalent streptavidin or bivalent immunoglobulin scaffolds. The peptide complex with streptavidin shows pronounced hepatotropism in vivo, replicating the biodistribution of full-length RAP.     

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLa^K44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy.     

The 39-kDa receptor-associated protein interacts with two members of the low density lipoprotein receptor family, alpha 2-macroglobulin receptor and glycoprotein 330.

The 39-kDa receptor-associated protein (RAP) binds to the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and inhibits binding of ligands to this receptor. The in vivo function of RAP may be to regulate ligand binding and/or assist in the correct biosynthetic processing or trafficking of the alpha 2MR/LRP. Here we show that RAP binds another putative receptor, the kidney glycoprotein 330 (gp330). Gp330 is a high molecular weight glycoprotein that is structurally similar to both the alpha 2MR/LRP and low density lipoprotein receptor. The ability of RAP to bind to gp330 was demonstrated by ligand blotting and solid phase binding assays, which showed that RAP binds to gp330 with high affinity (Kd = 8 nM). Exploiting the interaction of gp330 and RAP, we purified gp330 by affinity chromatography with a column of RAP coupled to Sepharose. Gp330 preparations obtained by this procedure were notably more homogeneous than those obtained by conventional methods. Immunocytochemical staining of human kidney sections localized RAP to the brush-border epithelium of proximal tubules. The fact that gp330 is also primarily expressed by proximal tubule epithelial cells strengthens the likelihood that the interaction between gp330 and RAP occurs in vivo. The functional significance of RAP binding to gp330 may be to antagonize ligand binding as has been demonstrated for the alpha 2MR/LRP or to assist in the biosynthetic processing and/or trafficking of this receptor.     

Molecular chaperone RAP interacts with LRP1 in a dynamic bivalent mode and enhances folding of ligand-binding regions of other LDLR family receptors

The low-density lipoprotein receptor (LDLR) family of receptors are cell-surface receptors that internalize numerous ligands and play crucial role in various processes, such as lipoprotein metabolism, hemostasis, fetal development, etc. Previously, receptor-associated protein (RAP) was described as a molecular chaperone for LDLR-related protein 1 (LRP1), a prominent member of the LDLR family. We aimed to verify this role of RAP for LRP1 and two other LDLR family receptors, LDLR and vLDLR, and to investigate the mechanisms of respective interactions using a cell culture model system, purified system, and in silico modelling. Upon coexpression of RAP with clusters of the ligand-binding complement repeats (CRs) of the receptors in secreted form in insect cells culture, the isolated proteins had increased yield, enhanced folding, and improved binding properties compared with proteins expressed without RAP, as determined by circular dichroism and surface plasmon resonance. Within LRP1 CR-clusters II and IV, we identified multiple sites comprised of adjacent CR doublets, which provide alternative bivalent binding combinations with specific pairs of lysines on RAP. Mutational analysis of these lysines within each of isolated RAP D1/D2 and D3 domains having high affinity to LRP1 and of conserved tryptophans on selected CR-doublets of LRP1, as well as in silico docking of a model LRP1 CR-triplet with RAP, indicated a universal role for these residues in interaction of RAP and LRP1. Consequently, we propose a new model of RAP interaction with LDLR family receptors based on switching of the bivalent contacts between molecules over time in a dynamic mode.     

Structural insights into recognition of β2‐glycoprotein I by the lipoprotein receptors

The interactions of β2 glycoprotein I (B2GPI) with the receptors of the low‐density lipoprotein receptor (LDLR) family are implicated in the clearance of negatively charged phospholipids and apoptotic cells and, in the presence of autoimmune anti‐B2GPI antibodies, in cell activation, which might play a role in the pathology of antiphospholipid syndrome (APS). The ligand‐binding domains of the lipoprotein receptors consist of multiple homologous LA modules connected by flexible linkers. In this study, we investigated at the atomic level the features of the LA modules required for binding to B2GPI. To compare the binding interface in B2GPI/LA complex to that observed in the high‐resolution co‐crystal structure of the receptor associated protein (RAP) with a pair of LA modules 3 and 4 from the LDLR, we used LA4 in our studies. Using solution NMR spectroscopy, we found that LA4 interacts with B2GPI and the binding site for B2GPI on the ¹⁵N‐labeled LA4 is formed by the calcium coordinating residues of the LA module. We built a model for the complex between domain V of B2GPI (B2GPI‐DV) and LA4 without introducing any experimentally derived constraints into the docking procedure. Our model, which is in the agreement with the NMR data, suggests that the binding interface of B2GPI for the lipoprotein receptors is centered at three lysine residues of B2GPI‐DV, Lys 308, Lys 282, and Lys317. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Dissection of RAP-LRP interactions: binding of RAP and RAP fragments to complement-like repeats 7 and 8 from ligand binding cluster II of LRP

The receptor associated protein (RAP) is a three domain 38kDa ER-resident chaperone that helps folding of LRP and other LDL receptor family members and prevents premature binding of protein ligands. It competes strongly with all known LRP ligands. To further understanding of the specificity of RAP-LRP interactions, the binding of RAP and RAP fragments to two domains (CR7-CR8) from one of the main ligand-binding regions of LRP has been examined by 2D HSQC NMR spectroscopy and isothermal titration calorimetry. We found that RAP contains two binding sites for CR7-CR8, with the higher affinity site (K(d) approximately 1microM) located in the C-terminal two-thirds and the weaker site (K(d) approximately 5microM) in the N-terminal third of RAP. Residues from both CR7 and CR8 are involved in binding at each RAP site. The presence of more than one binding site on RAP for CR domains from LRP, together with the previous demonstration by others that RAP can bind to CR5-CR6 with comparably low affinities suggest an explanation for the dual roles of RAP as a folding chaperone and a tight competitive inhibitor of ligand binding.     

Structural organization of the receptor associated protein

The receptor associated protein (RAP) is a 38 kDa ER-resident protein that binds tightly to the low density lipoprotein receptor-related protein (LRP), and other members of the LDL receptor family of receptors, and competes with all known LRP ligands for binding to LRP. To better understand the domain structure and organization of RAP, we have expressed RAP subfragments and examined them by two-dimensional HSQC NMR and fluorescence spectroscopies, by differential scanning calorimetry, and by both equilibrium and velocity sedimentation measurements. We found that the protein is organized into three domains located in the first third (1D), middle third (2D), and last third (3D) of the protein. All three domains adopt stable tertiary structure as isolated domains and are monomers. Whereas domains 1D and 2D do not interact with one another, 3D interacts with 2D, both in a 2D-3D construct and in intact RAP. Sedimentation measurements also indicated that intact RAP, although monomeric, is significantly elongated.     

The single ligand-binding repeat of Tva, a low density lipoprotein receptor-related protein, contains two ligand-binding surfaces

The receptor for avian sarcoma/leukosis virus subtype A (ASLV-A), Tva, is the simplest member of the low density lipoprotein receptor family containing a single ligand-binding repeat (LBR). Most LBRs contain a central Trp (Trp33 in Tva) that is important for ligand binding and, for the low density lipoprotein receptor, is associated with familial hypercholesterolemia. The Tva ligand-binding module contains a second Trp (Trp48) that is part of a DEW motif present in a subset of LBRs. Trp48 is important for ASLV-A infectivity. A soluble Tva (sTva) ligand-binding module is sufficient for ASLV-A infectivity. Tva interacts with the viral glycoprotein, and a soluble receptor-binding domain (SUA) binds sTva with picomolar affinity. We investigated whether Tva, a retroviral receptor, could behave as a classic LBR by assessing sTva interactions with the universal receptor-associated protein (RAP) and comparing these interactions with those between sTva and its viral ligand (SUA). To address the role of the two Trp residues in Tva function, we prepared sTva harboring mutations of Trp33, Trp48, or both and determined the binding kinetics with RAP and SUA. We found that sTva behaved as a "normal" receptor toward RAP, requiring both calcium and Trp33 for binding. However, sTva binding to SUA required neither calcium nor Trp33. Furthermore, sTva could bind both RAP and SUA simultaneously. These results show that the single LBR of Tva has two ligand-binding sites, raising the possibility that other LBRs may also.     

Allosteric modulation of ligand binding to low density lipoprotein receptor-related protein by the receptor-associated protein requires critical lysine residues within its carboxyl-terminal domain

The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that recognizes more than 30 different ligands and plays important roles in protease and lipoprotein catabolism. Ligand binding to newly synthesized LRP is modulated by the receptor-associated protein (RAP), an endoplasmic reticulum-resident protein that functions as a molecular chaperone and prevents ligands from associating with LRP via an allosteric-type mechanism. RAP is a multidomain protein that contains two independent LRP binding sites, one located at the amino-terminal portion of the molecule and the other at the carboxyl-terminal portion of the molecule. The objective of the present investigation was to gain insight into how these two regions of RAP interact with LRP and function to modulate its ligand binding properties. These objectives were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the carboxyl-terminal domain that are necessary for binding of this region of RAP to LRP and to heparin. RAP molecules in which either of these two lysine residues was mutated still bound LRP but with reduced affinity. Furthermore, the mutant RAPs were significantly impaired in their ability to inhibit alpha(2)M* binding to LRP via allosteric mechanisms. In contrast, the mutant RAP molecules were still effective at inhibiting uPA.PAI-1 binding to LRP. These results confirm that both LRP binding sites within RAP cooperate to inhibit ligand binding via an allosteric mechanism.     

Endocytic trafficking of megalin/RAP complexes: dissociation of the complexes in late endosomes.

Megalin (gp330) is a member of the low-density lipoprotein receptor gene family. Like other members of the family, it is an endocytic receptor that binds a number of specific ligands. Megalin also binds the receptor-associated protein (RAP) that serves as an exocytic traffic chaperone and inhibits ligand binding to the receptor. To investigate the fate of megalin/RAP complexes, we bound RAP glutathione-S-transferase fusion protein (RAP-GST) to megalin at the surface of L2 yolk sac carcinoma cells and followed the trafficking of the complexes by immunofluorescence and immunogold labeling and by their distribution on Percoll gradients. We show that megalin/RAP-GST complexes, which are internalized via clathrin-coated pits, are delivered to early endosomes where they accumulate during an 18 degrees C temperature block and colocalize with transferrin and transferrin receptor. Upon release from the temperature block, the complexes travel to late endosomes where they colocalize with rab7 and can be coprecipitated with anti-RAP-GST antibodies. Dissociation of the complex occurs in late endosomes and is most likely triggered by the low pH (approximately 5.5) of this compartment. RAP is then rapidly delivered to lysosomes and degraded whereas megalin is recycled to the cell surface. When the ligand, lipoprotein lipase, was bound to megalin, the receptor was found to recycle through early endosomes. We conclude that in contrast to receptor/ligand complexes, megalin/RAP complexes traffic through late endosomes, which is a novelty for members of the low-density lipoprotein receptor gene family.     

A novel mechanism for controlling the activity of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Multiple regulatory sites for 39-kDa receptor-associated protein.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) consists of two polypeptides, 515 and 85 kDa, that are noncovalently associated. A 39-kDa polypeptide, termed the receptor-associated protein (RAP), interacts with the 515-kDa subunit after biosynthesis of these molecules and remains associated on the cell surface. This molecule regulates ligand binding of alpha 2MR/LRP (Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S. (1991) J. Biol. Chem. 266, 21232-21238). Titration and binding studies indicate that RAP binds to two equivalent binding sites on alpha 2MR/LRP, with a KD of 14 nM. Heterologous ligand displacement experiments demonstrated that RAP completely inhibits the binding of 125I-activated alpha 2M to human fibroblasts and to the purified alpha 2MR/LRP, with a Ki of 23 and 26 nM, respectively. A direct correlation between the degree of binding of RAP to the receptor and the degree of ligand inhibition was observed, indicating that as the RAP binding sites are saturated, alpha 2MR/LRP loses its ability to bind ligands. Thus, the amount of RAP bound to alpha 2MR/LRP dictates the level of receptor activity. A model is proposed in which alpha 2MR/LRP contains multiple ligand binding sites, each regulated by a separate RAP site.     

Hsp70-RAP46 interaction in downregulation of DNA binding by glucocorticoid receptor

Receptor-associating protein 46 (RAP46) is a cochaperone that regulates the transactivation function of several steroid receptors. It is transported into the nucleus by a liganded glucocorticoid receptor where it downregulates DNA binding and transactivation by this receptor. The N- and C-termini of RAP46 are both implicated in its negative regulatory function. In metabolic labelling experiments, we have shown that the N-terminus of RAP46 is modified by phosphorylation, but this does not contribute to the downregulation of glucocorticoid receptor activity. However, deletion of a sequence that binds 70 kDa heat shock protein (Hsp70) and the constitutive isoform of Hsp70 (Hsc70) at the C-terminus of RAP46 abrogated its negative regulatory action. Surface plasmon resonance studies showed that RAP46 binds the glucocorticoid receptor only when it has interacted with Hsp70/Hsc70, and confocal immunofluorescence analyses revealed a nuclear transport of Hsp70/Hsc70 by the liganded receptor. Together these findings demonstrate an important contribution of Hsp70/Hsc70 in the binding of RAP46 to the glucocorticoid receptor and suggest a role for this molecular chaperone in the RAP46-mediated downregulation of glucocorticoid receptor activity.     

Gene transfer into human hepatoma cells by receptor-associated protein/polylysine conjugates

Receptor-associated protein (RAP) is a ligand for all members of low-density lipoprotein (LDL) receptor families. RAP is internalized into cells via receptor-mediated endocytic trafficking, making it an attractive mechanism for efficient gene delivery. In this study, we have developed a gene delivery system using RAP as a targeting ligand. A RAP cDNA lacking a C-terminal heparin-binding domain was amplified by polymerase chain reaction (PCR) from a human liver cDNA library and was reamplified by using a primer containing a cysteine codon at its carboxyl end to facilitate its conjugation to polylysine (polyK). RAP was purified using a bacterial expression system and coupled to poly-D-lysine (PDL) or poly-L-lysine (PLL) of average MW 50 kDa via the heterobifunctional cross-linker SPDP. Using fluorescence-labeled RAP ligand, cellular uptake of the transfection complexes into HepG2 cells was shown to be highly efficient and more specific to PDL-conjugated RAP compared with PLL-conjugated one. Plasmid DNA containing a luciferase reporter gene was condensed with either RAP-PDL or RAP-PLL. In vitro transfection into HepG2 cells with RAP-PDL conjugate resulted in significantly higher luciferase expression levels in comparison to either nonconjugated PDL, or RAP-PLL, or LipofecAMINE/DNA complexes in the presence of 10% fetal bovine serum. Luciferase expression was inhibited by the addition of excess RAP. Treatment of the cells with Lovastatin, which inhibits HMG-Co reductase and increases expression of LDL receptor, stimulates luciferase expression, suggesting that the gene delivery is specifically mediated by LDL receptor. Thus, RAP-PDL conjugates have the potential to be used as a new nonviral gene delivery vector.     

Intrinsic lattice formation by the ryanodine receptor calcium-release channel

Recent theoretical analysis of a model lattice of interacting transmembrane receptor proteins has indicated that such clustering in the membrane could provide a novel mechanism for regulating receptor signalling in cells. It has been calculated that cooperative interactions between receptors organized into a cluster, or array, in the membrane would dramatically increase their sensitivity to activation by ligand. Sensitivity to ligand would increase with the extent of spread of activity within the receptor lattice. Hence, formation of extensive receptor lattices in the membrane would allow a large population of receptors to be simultaneously switched on, or off, by a very small change in ligand concentration. We show here that lattice formation is an intrinsic property of an integral membrane protein, the ryanodine-sensitive calcium-release channel (RyR) of endoplasmic reticulum. The purified protein spontaneously assembled into two-dimensional lattices in solution, enabling the construction of a 25 A projection map that identifies the mode of interaction between RyR oligomers. Our observations on the RyR provide a new perspective on various properties of cell signalling via this and other receptors.     

Interactions of vasopressin and oxytocin receptors with vasopressin analogues substituted in position 2 with 3,3′‐diphenylalanine – a molecular docking study

Vasopressin and oxytocin receptors belong to the superfamily of G protein‐coupled receptors and play an important role in many physiological functions. They are also involved in a number of pathological conditions being important drug targets. In this work, four vasopressin analogues substituted at position 2 with 3,3′‐diphenylalanine have been docked into partially flexible vasopressin and oxytocin receptors. The bulky residue at position 2 acts as a structural restraint much stronger in the oxytocin receptor (OTR) than in the vasopressin V2 receptor (V2R), resulting in a different location of the analogues in these receptors. This explains the different, either agonistic or antagonistic, activities of the analogues in V2R and OTR, respectively. In all complexes, the conserved polar residues serve as anchor points for the ligand both in OTR and V2R. Strong interactions of the C‐terminus of analogue II ([Mpa¹,d ‐Dpa²,Val⁴,d ‐Arg⁸]VP) with extracellular loop 3 may be responsible for its highest activity at V2R. It also appears that V2R adapts more readily to the docking analogues by conformational changes in the aromatic side chains triggering receptor activation. A weak activity at V1a vasopressin receptor appears to be caused by weak receptor–ligand interactions. Results of this study may facilitate a rational design of new analogues with the highest activity/selectivity at vasopressin and OTRs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.