Laurence J Miller

Mayo Clinic - Scottsdale, Scottsdale, Arizona, United States

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Publications (234)1198.92 Total impact

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    ABSTRACT: Amino-terminal regions of secretin-family peptides contain key determinants for biological activity and binding specificity, although the nature of interactions with receptors is unclear. A helix N-capping motif within this region has been postulated to directly contribute to agonist activity, while also stabilizing formation of a helix extending toward the peptide carboxyl terminus and docking within the receptor amino terminus. We use cysteine trapping to systematically explore spatial approximations between cysteines replacing each residue in this motif of secretin, Phe(6), Thr(7) and Leu(10), and cysteines incorporated into the extracellular face of the receptor. Each peptide was a full agonist for cAMP, although having lower binding affinity than natural hormone. These bound to COS cells expressing 61 receptor constructs incorporating cysteines in every position along each extracellular loop (ECL) and adjacent parts of transmembrane (TM) segments. Patterns of covalent labeling were distinct for each probe, with Cys(6)-sec labeling multiple residues in the carboxyl-terminal half of ECL2 and throughout ECL3, Cys(7)-sec predominantly labeled only single residues in the carboxyl-terminal end of ECL2 and the amino-terminal end of ECL3, and Cys(10)-sec not efficiently labeling any of these residues. These spatial constraints were used to refine our model of secretin bound to its receptor, now bringing ECL3 above the amino terminus of the ligand, and revealing possible charge-charge interactions between this part of secretin and receptor residues in TM5, TM6, ECL2, and ECL3, which can orient and stabilize the peptide-receptor complex. This was validated by testing predicted approximations by mutagenesis and residue-residue complementation studies.
    Full-text · Article · Jan 2016 · Journal of Biological Chemistry
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    ABSTRACT: The glucagon-like peptide 1 (GLP-1) receptor is a class B G protein-coupled receptor (GPCR) that is a key target for treatments for type II diabetes and obesity. This receptor, like other class B GPCRs, displays biased agonism, though the physiological significance of this is yet to be elucidated. Previous work has implicated R2.60(190), N3.43(240), Q7.49(394) and H6.52(363) as key residues involved in peptide-mediated biased agonism (Wootten et al., 2013a), with R2.60(190), N3.43(240) and Q7.49(394) predicted to form a polar interaction network. In this study, we used novel insight gained from recent crystal structures of the transmembrane domains of the glucagon and corticotropin releasing factor 1 (CRF1) receptors to develop improved models of the GLP-1 receptor that predict additional key molecular interactions with these amino acids. We have introduced E6.53(364)A, N3.43(240)Q, Q7.49(394)N and N3.43(240)Q/ Q7.49(394)N mutations to probe the role of predicted H-bonding and charge-charge interactions in driving cAMP, calcium or ERK signaling. A polar interaction between E6.53(364) and R2.60(190) was predicted to be important for GLP-1- and exendin-4-, but not oxyntomodulin-mediated cAMP formation and also ERK1/2 phosphorylation. In contrast, Q7.49(394), but not R2.60(190)/ E6.53(364) was critical for calcium mobilisation for all three peptides. Mutation of N3.43(240) and Q7.49(394) had differential effects on individual peptides providing evidence for molecular differences in activation transition. Collectively, this work expands our understanding of peptide-mediated signaling from the GLP-1 receptor and the key role that the central polar network plays in these events.
    Full-text · Article · Dec 2015 · Molecular pharmacology
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    ABSTRACT: The type 1 cholecystokinin receptor (CCK1R) has multiple physiologic roles relating to nutrient homeostasis, including mediation of postcibal satiety. This effect has been central in efforts to develop agonists of this receptor as part of a program to manage and/or prevent obesity. While a number of small molecule CCK1R agonists have been developed, none have yet been approved for clinical use, based on inadequate efficacy, side effects, or the potential for toxicity. Understanding the molecular details of docking and mechanism of action of these ligands can be helpful in the rational refinement and enhancement of small molecule drug candidates. In the current work, we have defined the mechanism of binding and activity of two triazolobenzodiazepinones, CE-326597 and PF-04756956, which are reported to be full agonist ligands. To achieve this, we utilized receptor binding with a series of allosteric and orthosteric radioligands at structurally related CCK1R and CCK2R, as well as chimeric CCK1R/CCK2R constructs exchanging residues in the allosteric pocket, and assessment of biological activity. These triazolobenzodiazepinones docked within the intramembranous small molecule allosteric ligand pocket, with higher affinity binding to CCK2R than CCK1R, yet with biological activity exclusive to or greatly enhanced at CCK1R. These ligands exhibited cooperativity with benzodiazepine binding across the CCK1R homodimeric complex, resulting in their ability to inhibit only a fraction of the saturable binding of a benzodiazepine radioligand, unlike other small molecule antagonists and agonists of this receptor. This may contribute to the understanding of the unique short duration and reversible gallbladder contraction observed in vivo upon administration of these drugs.
    No preview · Article · Dec 2015 · Journal of Medicinal Chemistry
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    ABSTRACT: Dysfunction of the type 1 cholecystokinin receptor (CCK1R) as an effect of increased gallbladder muscularis membrane cholesterol has been implicated in the pathogenesis of cholesterol gallstones. Administration of the ursodeoxycholic acid, which is structurally related to cholesterol, has been shown to have beneficial effects on gallstone formation. Our aims were to explore possible direct effects and mechanism of action of bile acids on CCK receptor function. We studied the effects of structurally-related hydrophobic chenodeoxycholic acid and hydrophilic ursodeoxycholic acid in vitro on CCK receptor function in the setting of normal and elevated membrane cholesterol. We also examined their effects on a cholesterol-insensitive CCK1R mutant (Y140A) disrupting a key site of cholesterol action. The results show that similar to impact of cholesterol on CCK receptors, bile acid effects were limited to CCK1R, with no effects on CCK2R. Chenodeoxycholic acid had negative impact on CCK1R function, while ursodeoxycholic acid had no effect on CCK1R function in normal membranes, while being protective against the negative impact of elevated cholesterol on this receptor. The cholesterol-insensitive CCK1R mutant, Y140A, was resistant to effects of both bile acids. These data suggest that bile acids compete with the action of cholesterol on CCK1R, probably by interacting at the same site, although the conformational impact of each bile acid appears to be different, with ursodeoxycholic acid capable of correcting the abnormal conformation of CCK1R in high cholesterol. This mechanism may contribute to its beneficial effect in reducing cholesterol gallstone formation. Copyright © 2015, American Journal of Physiology- Gastrointestinal and Liver Physiology.
    Full-text · Article · Jul 2015 · AJP Gastrointestinal and Liver Physiology
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    Aditya J Desai · Brad R Henke · Laurence J Miller
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    ABSTRACT: Cholecystokinin (CCK) acts at the type 1 cholecystokinin receptor (CCK1R) to elicit satiety and is a well-established drug target for obesity. To date, small molecule agonists have been developed, but have failed to demonstrate adequate efficacy in clinical trials, and concerns about side effects and potential toxicity have limited further development of full agonists. The use of positive allosteric modulators (PAMs) without intrinsic agonist activity that are active only for a brief period of time after a meal might represent a safer alternative. Here, we propose a possible novel strategy to develop such compounds by modifying the agonist 'trigger' of an existing small molecule agonist. We have studied analogues of the 1,5-benzodiazepine agonist, GI181771X, in which the N1-isopropyl agonist 'trigger' was modified. While agonist activity was greatly reduced in these compounds, they acted as negative, rather than positive modulators. The parent drug was also found to exhibit no positive modulation of CCK action. Receptor structure-activity relationship studies demonstrated that the mode of docking these derivatives was distinct from that of the parent compound, perhaps explaining their action as negative allosteric modulators. We conclude that this outcome is likely characteristic of the parental agonist, and that this strategy may be more successfully utilized with a parental ago-PAM, possessing intrinsic positive modulatory activity. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Full-text · Article · Mar 2015 · Bioorganic & medicinal chemistry letters
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    ABSTRACT: The glucagon-like peptide-1 receptor (GLP-1R) is a Class B G protein-coupled receptor (GPCR) that has a critical role in the regulation of glucose homeostasis, principally though regulation of insulin secretion. The receptor system is highly complex, with the ability to be activated by both endogenous (GLP-1(1-36)NH2, GLP-1(1-37), GLP-1(7-36)NH2, GLP-1(7-37), oxyntomodulin) and exogenous (exendin-4) peptides in addition to small molecule allosteric agonists (6,7-dichloro-2-methylsulfonyl-3-tert-butylaminoquinoxaline (compound 2), 4-(3-benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP)). Furthermore, the GLP-1R is subject to single nucleotide polymorphic variance, resulting in amino acid changes in the receptor protein. In this study, we investigated two polymorphic variants that have previously been reported to impact peptide-mediated receptor activity (M149) and small molecule allostery (C333). These residues were mutated to a series of alternate amino acids and their functionality monitored across physiologically significant signaling pathways including cAMP, extracellular signal-regulated kinase 1 and 2 phosphorylation (pERK1/2) and intracellular Ca2+ mobilization, in addition to peptide binding and cell surface expression. We observed that residue 149 is highly sensitive to mutation, with almost all peptide responses significantly attenuated at mutated receptors. However, most reductions in activity were able to be restored by the small molecule allosteric agonist, compound 2. Conversely, mutation of residue 333 has little impact on peptide-mediated receptor activation, but this activity is unable to be modulated by compound 2 to the same extent as that observed at the wildtype receptor. These results provide insight into the importance of residues 149 and 333 in peptide function as well as highlighting the complexities of allosteric modulation within this receptor system. The American Society for Pharmacology and Experimental Therapeutics.
    No preview · Article · Jan 2015 · Journal of Pharmacology and Experimental Therapeutics
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    ABSTRACT: Understanding the molecular basis of ligand binding to receptors provides insights useful for rational drug design. This work describes development of a new antagonist radioligand (T-0632) of the type 1 cholecystokinin receptor (CCK1R) and exploration of the molecular basis of its binding. This radioligand bound specifically with high affinity within an allosteric pocket of CCK1R. T-0632 fully inhibited binding and action of CCK at this receptor, while exhibiting no saturable binding to closely-related CCK2R. Chimeric CCK1R/CCK2R constructs were used to explore the molecular basis of T-0632 binding. Exchanging exonic regions revealed functional importance of CCK1R exon 3, extending from bottom of TM3 to top of TM5, including portions of the intramembranous pocket as well as ECL2. However, CCK1R mutants in which each residue facing the pocket was changed to that present in CCK2R had no negative impact on T-0632 binding. Extending the chimeric approach to ECL2 established importance of its carboxyl-terminal region, and site-directed mutagenesis of each non-conserved residue in this region revealed importance of Ser(208) at the top of TM5. A molecular model of T-0632-occupied CCK1R was consistent with these experimental determinants, also identifying Met(121) in TM3 and Arg(336) in TM6 as important. While these residues are conserved in CCK2R, mutating them had distinct impact on the two closely-related receptors, suggesting differential orientation. This establishes the molecular basis of binding a highly selective non-peptidyl allosteric antagonist of CCK1R, illustrating differences in docking that extend beyond determinants attributable to distinct residues lining the intramembranous pocket in the two receptor subtypes.
    No preview · Article · Oct 2014 · Molecular pharmacology
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    Full-text · Dataset · Aug 2014
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    Aditya J Desai · Kaleeckal G Harikumar · Laurence J Miller
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    ABSTRACT: Cholecystokinin (CCK) stimulates the type 1 CCK receptor (CCK1R) to elicit satiety after a meal. Agonists with this activity, while potentially useful for treatment of obesity, also can have side effects and toxicities of concern, making development of an intrinsically inactive positive allosteric modulator (PAM) quite attractive. PAMs also have the potential to correct the defective receptor-G protein coupling observed in the high membrane cholesterol environment described in metabolic syndrome. Current model systems to study CCK1R in such an environment are unstable and expensive to maintain. We now report that the Y140A mutation within a cholesterol-binding motif and the conserved, class A GPCR-specific E/DRY signature sequence results in ligand binding and activity characteristics similar to wild type CCK1R in a high cholesterol environment. This is true for natural CCK, as well as ligands with distinct chemistries and activity profiles. Additionally, the Y140A construct also behaved like CCK1R in high cholesterol in regard to its internalization, sensitivity to a non-hydrolyzable GTP analogue, and anisotropy of a bound fluorescent CCK analogue. Chimeric CCK1R/CCK2R constructs that systematically changed the residues in the allosteric ligand-binding pocket were studied in the presence of Y140A. This established increased importance of unique residues within TM3 and reduced importance of TM2 for binding in the presence of this mutation, with the agonist trigger likely pulled away from its Leu(356) target on TM7. The distinct conformation of this intramembranous pocket within Y140A CCK1R provides an opportunity to normalize this using a small molecule allosteric ligand, thereby providing safe and effective correction of the coupling defect in metabolic syndrome.
    Full-text · Article · May 2014 · Journal of Biological Chemistry
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    ABSTRACT: Angiotensin (ANGII) and secretin (SCT) share overlapping, interdependent osmoregulatory functions in brain, where SCT peptide/receptor function is required for ANGII action, yet the molecular basis is unknown. Since receptors for these peptides (AT1aR, SCTR) are coexpressed in osmoregulatory centers, a possible mechanism is formation of a cross-class receptor heterocomplex. Here, we demonstrate such a complex and its functional importance to modulate signaling. Association of AT1aR with SCTR reduced ability of SCT to stimulate cyclic adenosine monophosphate (cAMP), with signaling augmented in presence of ANGII or constitutively active AT1aR. Several transmembrane (TM) peptides of these receptors were able to affect their conformation within complexes, reducing receptor BRET signals. AT1aR TM1 affected only formation and activity of the heterocomplex, without effect on homomers of either receptor, and reduced SCT-stimulated cAMP responses in cells expressing both receptors. This peptide was active in vivo by injection into mouse lateral ventricle, thereby suppressing water-drinking behavior after hyperosmotic shock, similar to SCTR knockouts. This supports the interpretation that active conformation of AT1aR is a key modulator of cAMP responses induced by SCT stimulation of SCTR. The SCTR/AT1aR complex is physiologically important, providing differential signaling to SCT in settings of hyperosmolality or food intake, modulated by differences in levels of ANGII.-Lee, L. T. O., Ng, S. Y. L., Chu, J. Y. S., Sekar, R., Harikumar, K. G., Miller, L. J., Chow, B. K. C. Transmembrane peptides as unique tools to demonstrate the in vivo action of a cross-class GPCR heterocomplex.
    Full-text · Article · Mar 2014 · The FASEB Journal
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    ABSTRACT: Norrin is a cysteine-rich growth factor that is required for angiogenesis in the eye, ear, brain, and female reproductive organs. It functions as an atypical Wnt ligand by specifically binding to the Frizzled 4 (Fz4) receptor. Here we report the crystal structure of Norrin, which reveals a unique dimeric structure with each monomer adopting a conserved cystine knot fold. Functional studies demonstrate that the novel Norrin dimer interface is required for Fz4 activation. Furthermore, we demonstrate that Norrin contains separate binding sites for Fz4 and for the Wnt ligand coreceptor Lrp5 (low-density lipoprotein-related protein 5) or Lrp6. Instead of inducing Fz4 dimerization, Norrin induces the formation of a ternary complex with Fz4 and Lrp5/6 by binding to their respective extracellular domains. These results provide crucial insights into the assembly and activation of the Norrin-Fz4-Lrp5/6 signaling complex.
    No preview · Article · Nov 2013 · Genes & development
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    ABSTRACT: The glucagon-like peptide-1 receptor (GLP-1R) controls the physiological responses to the incretin hormone glucagon-like peptide-1 and is a major therapeutic target for the treatment of type 2 diabetes, owing to the broad range of effects that are mediated upon its activation. These include the promotion of glucose-dependent insulin secretion, increased insulin biosynthesis, preservation of β-cell mass, improved peripheral insulin action, and promotion of weight loss. Regulation of GLP-1R function is complex, with multiple endogenous and exogenous peptides that interact with the receptor that result in the activation of numerous downstream signaling cascades. The current understanding of GLP-1R signaling and regulation is limited, with the desired spectrum of signaling required for the ideal therapeutic outcome still to be determined. In addition, there are several single-nucleotide polymorphisms (used in this review as defining a natural change of single nucleotide in the receptor sequence; clinically, this is viewed as a single-nucleotide polymorphism only if the frequency of the mutation occurs in 1% or more of the population) distributed within the coding sequence of the receptor protein that have the potential to produce differential responses for distinct ligands. In this review, we discuss the current understanding of GLP-1R function, in particular highlighting recent advances in the field on ligand-directed signal bias, allosteric modulation, and probe dependence and the implications of these behaviors for drug discovery and development.
    No preview · Article · Jul 2013 · Molecular Endocrinology
  • Maoqing Dong · Laurence J Miller
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    ABSTRACT: 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 analogue probes with sites of covalent attachment extending from position 26 through 32 were characterized, with the highest affinity analogues 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 Phe(120) in the first extracellular loop. This was in contrast to its covalent attachment to Glu(345) in the third extracellular loop of the type 1 CCK receptor, directly documenting differences in mode of docking this peptide to these receptors.
    No preview · Article · Jun 2013 · Peptides
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    ABSTRACT: Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally-related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu7.39 that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a trigger for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an AUC value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (AUC 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu7.39, while the role of this residue was less clear for chemically-distinct agonists.
    Full-text · Article · Jun 2013 · Journal of Biological Chemistry
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    ABSTRACT: The function of Kruppel-like Factor 11 (KLF11) in the regulation of metabolic pathways is conserved from flies to human. Alterations in KLF11 function result in Maturity Onset Diabetes of the Young 7 (MODY7) and neonatal diabetes, however, the mechanisms underlying the role of this protein in metabolic disorders remain unclear. Here, we seek to investigate how the A347S genetic variant, present in MODY7 patients, modulates KLF11 transcriptional activity. A347S affects a previously identified transcriptional regulatory domain 3 (TRD3) for which co-regulators remain unknown. Structure-oriented sequence analyses, described here, predict that the KLF11 TRD3 represents an evolutionarily conserved protein domain. Combined, yeast two-hybrid and protein arrays demonstrate that the TRD3 binds WD40, WWI, WWII, and SH3-domain containing proteins. Using one of these proteins as a model, guanine nucleotide binding protein beta 2 (Gβ2), we investigate the functional consequences of KLF11 coupling to a TRD3 binding partner. Combined, immunoprecipitation and biomolecular fluorescence complementation assays confirm that activation of three different metabolic GPCR receptors (β-adrenergic, secretin and CCK) induce nuclear translocation of Gβ2 to directly bind KLF11 in a manner that is disrupted by the MODY7 A347S variant. Using genome-wide expression profiles, we identify metabolic gene networks impacted upon TRD3 disruption. Furthermore, A347S disrupts KLF11-mediated increases in basal insulin levels and promoter activity, combined with blunted glucose-stimulated insulin secretion. Thus, this study characterizes a novel protein-protein interaction domain disrupted in a KLF gene variant that associates to MODY7, contributing to our understanding of gene regulation events in complex metabolic diseases.
    No preview · Article · Apr 2013 · Journal of Biological Chemistry
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    Laurence J Miller

    Preview · Article · Apr 2013 · Molecular Metabolism
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    ABSTRACT: Recently, the concept of ligand-directed signaling-the ability of different ligands of an individual receptor to promote distinct patterns of cellular response-has gained much traction in the field of drug discovery, with the potential to sculpt biological response to favor therapeutically beneficial signaling pathways over those leading to harmful effects. However, there is limited understanding of the mechanistic basis underlying biased signaling. The glucagon-like peptide-1 receptor is a major target for treatment of type-2 diabetes and is subject to ligand-directed signaling. Here, we demonstrate the importance of polar transmembrane residues conserved within family B G protein-coupled receptors, not only for protein folding and expression, but also in controlling activation transition, ligand-biased, and pathway-biased signaling. Distinct clusters of polar residues were important for receptor activation and signal preference, globally changing the profile of receptor response to distinct peptide ligands, including endogenous ligands glucagon-like peptide-1, oxyntomodulin, and the clinically used mimetic exendin-4.
    Full-text · Article · Mar 2013 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Type 2 diabetes is a major global health problem and there is ongoing research for new treatments to manage the disease. The GLP-1R (glucagon-like peptide-1 receptor) controls the physiological response to the incretin peptide, GLP-1, and is currently a major target for the development of therapeutics owing to the broad range of potential beneficial effects in Type 2 diabetes. These include promotion of glucose-dependent insulin secretion, increased insulin biosynthesis, preservation of β-cell mass, improved peripheral insulin sensitivity and promotion of weight loss. Despite this, our understanding of GLP-1R function is still limited, with the desired spectrum of GLP-1R-mediated signalling yet to be determined. We review the current understanding of GLP-1R function, in particular, highlighting recent contributions in the field on allosteric modulation, probe-dependence and ligand-directed signal bias and how these behaviours may influence future drug development.
    Full-text · Article · Feb 2013 · Biochemical Society Transactions
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    Laurence J Miller · Maoqing Dong
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    ABSTRACT: Class B GPCRs (G-protein-coupled receptors) share heptahelical topology and G-protein binding with other superfamily members, yet have unique structures and modes of activation. Natural ligands for these receptors are moderate-length peptides with C-terminal α-helices. NMR and crystal structures of the peptide-bound disulfide-bonded receptor N-terminal domains demonstrate that these helices occupy a conserved groove; however, the details of this interaction vary from one receptor to another. In this review, we focus on the prototypic secretin receptor and use extensive intrinsic photoaffinity labelling, structure-activity series, alanine-replacement mutagenesis and fluorescence analysis to define the molecular basis for this interaction. Additionally, experimental validation of predictions coming from in silico molecular modelling has provided a basis for enhancement of binding affinity. Such insights will be useful in the rational development of drugs acting at this important group of targets.
    Preview · Article · Feb 2013 · Biochemical Society Transactions
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    ABSTRACT: The lipid microenvironment of membrane proteins can affect their structure, function, and regulation. We recently described differential effects of acute modification of membrane cholesterol on the function of type 1 and 2 cholecystokinin (CCK) receptors. We now explore the regulatory impact of chronic cholesterol modification on these receptors using novel receptor-bearing cell lines with elevated membrane cholesterol. Stable CCK1R and CCK2R expression was established in clonal lines of 25RA cells having gain-of-function in SCAP [sterol regulatory element binding protein (SREBP) cleavage-activating protein] and SRD15 cells having deficiencies in Insig-1 and Insig-2 enzymes affecting HMG CoA reductase and SREBP. Increased cholesterol in the plasma membrane of these cells was directly demonstrated, and receptor binding and signaling characteristics were shown to reflect predicted effects on receptor function. In both environments, both types of CCK receptors were internalized and recycled normally in response to agonist occupation. No differences in receptor distribution within the membrane were appreciated at the light microscopic level in these CHO-derived cell lines. Fluorescence anisotropy was studied for these receptors occupied by fluorescent agonist and antagonist, as well as when tagged with YFP. These studies demonstrated increased anisotropy of the agonist ligand occupying the active state of the CCK1R in a cholesterol-enriched environment, mimicking fluorescence of the uncoupled, inactive state of this receptor, while there was no effect of increasing cholesterol on fluorescence at the CCK2R. These cell lines should be quite useful for examining the functional characteristics of potential drugs that might be used in an abnormal lipid environment.
    No preview · Article · Jan 2013 · Lipids

Publication Stats

5k Citations
1,198.92 Total Impact Points

Institutions

  • 2004-2015
    • Mayo Clinic - Scottsdale
      Scottsdale, Arizona, United States
    • Mayo Foundation for Medical Education and Research
      • Department of Molecular Pharmacology and Experimental Therapeutics
      Рочестер, Michigan, United States
  • 2014
    • The University of Hong Kong
      • School of Biological Sciences
      Hong Kong, Hong Kong
  • 2013
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
    • Stanford Medicine
      Stanford, California, United States
  • 2008-2011
    • Monash University (Australia)
      • Department of Pharmacology
      Melbourne, Victoria, Australia
  • 1979-2010
    • Mayo Clinic - Rochester
      Rochester, Minnesota, United States
  • 2009
    • Universität Bern
      • Institute of Pathology
      Berna, Bern, Switzerland
  • 2006
    • Ehime University
      Matuyama, Ehime, Japan
    • University of Melbourne
      Melbourne, Victoria, Australia
  • 2002-2006
    • Vanderbilt University
      Nashville, Michigan, United States
    • Humboldt-Universität zu Berlin
      Berlín, Berlin, Germany
  • 2005
    • Universität Heidelberg
      Heidelburg, Baden-Württemberg, Germany
  • 2003
    • Moffitt Cancer Center
      Tampa, Florida, United States
  • 2001
    • University of Naples Federico II
      Napoli, Campania, Italy
  • 1998-1999
    • University of Washington Seattle
      Seattle, Washington, United States
  • 1992
    • University of Toronto
      • Department of Medicine
      Toronto, Ontario, Canada