Residues In Intracellular Loop Research Articles

Ubiquitin links smoothened to intraflagellar transport to regulate Hedgehog signaling.

In the absence of Hedgehog ligand, patched-1 (Ptch1) localizes to cilia and prevents ciliary accumulation and activation of smoothened (Smo). Upon ligand binding, Ptch1 is removed from cilia, and Smo is derepressed and accumulates in cilia where it activates signaling. The mechanisms regulating these dynamic movements are not well understood, but defects in intraflagellar transport components, including Ift27 and the BBSome, cause Smo to accumulate in cilia without pathway activation. We find that in the absence of ligand-induced pathway activation, Smo is ubiquitinated and removed from cilia, and this process is dependent on Ift27 and BBSome components. Activation of Hedgehog signaling decreases Smo ubiquitination and ciliary removal, resulting in its accumulation. Blocking ubiquitination of Smo by an E1 ligase inhibitor or by mutating two lysine residues in intracellular loop three causes Smo to aberrantly accumulate in cilia without pathway activation. These data provide a mechanism to control Smo's ciliary level during Hedgehog signaling by regulating the ubiquitination state of the receptor.

Lipid-Protein Interactions Are a Unique Property and Defining Feature of G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are membrane-bound proteins that depend on their lipid environment to carry out their physiological function. Combined efforts from many theoretical and experimental studies on the lipid-protein interaction profile of several GPCRs hint at an intricate relationship of these receptors with their surrounding membrane environment, with several lipids emerging as particularly important. Using coarse-grained molecular dynamics simulations, we explore the lipid-protein interaction profiles of 28 different GPCRs, spanning different levels of classification and conformational states and totaling to 1 ms of simulation time. We find a close relationship with lipids for all GPCRs simulated, in particular, cholesterol and phosphatidylinositol phosphate (PIP) lipids, but the number, location, and estimated strength of these interactions is dependent on the specific GPCR as well as its conformational state. Although both cholesterol and PIP lipids bind specifically to GPCRs, they utilize distinct mechanisms. Interactions with PIP lipids are mediated by charge-charge interactions with intracellular loop residues and stabilized by one or both of the transmembrane helices linked by the loop. Interactions with cholesterol, on the other hand, are mediated by a hydrophobic environment, usually made up of residues from more than one helix, capable of accommodating its ring structure and stabilized by interactions with aromatic and charged/polar residues. Cholesterol binding to GPCRs occurs in a small number of sites, some of which (like the binding site on the extracellular side of transmembrane 6/7) are shared among many class A GPCRs. Combined with a thorough investigation of the local membrane structure, our results provide a detailed picture of GPCR-lipid interactions. Additionally, we provide an accompanying website to interactively explore the lipid-protein interaction profile of all GPCRs simulated to facilitate analysis and comparison of our data.

Trafficking and other regulatory mechanisms for organic anion transporting polypeptides and organic anion transporters that modulate cellular drug and xenobiotic influx and that are dysregulated in disease.

Organic anion transporters (OATs) and organic anion-transporting polypeptides (OATPs), encoded by a number of solute carrier (SLC)22A and SLC organic anion (SLCO) genes, mediate the absorption and distribution of drugs and other xenobiotics. The regulation of OATs and OATPs is complex, comprising both transcriptional and post-translational mechanisms. Plasma membrane expression is required for cellular substrate influx by OATs/OATPs. Thus, interest in post-translational regulatory processes, including membrane targeting, endocytosis, recycling and degradation of transporter proteins, is increasing because these are critical for plasma membrane expression. After being synthesized, transporters undergo N-glycosylation in the endoplasmic reticulum and Golgi apparatus and are delivered to the plasma membrane by vesicular transport. Their expression at the cell surface is maintained by de novo synthesis and recycling, which occurs after clathrin- and/or caveolin-dependent endocytosis of existing protein. Several studies have shown that phosphorylation by signalling kinases is important for the internalization and recycling processes, although the transporter protein does not appear to be directly phosphorylated. After internalization, transporters that are targeted for degradation undergo ubiquitination, most likely on intracellular loop residues. Epigenetic mechanisms, including methylation of gene regulatory regions and transcription from alternate promoters, are also significant in the regulation of certain SLC22A/SLCO genes. The membrane expression of OATs/OATPs is dysregulated in disease, which affects drug efficacy and detoxification. Several transporters are expressed in the cytoplasmic subcompartment in disease states, which suggests that membrane targeting/internalization/recycling may be impaired. This article focuses on recent developments in OAT and OATP regulation, their dysregulation in disease and the significance for drug therapy.

Functional importance of two conserved residues in intracellular loop 1 and transmembrane region 2 of Family A GPCRs: Insights from ligand binding and signal transduction responses of D1 and D5 dopaminergic receptor mutants

For many G protein-coupled receptors (GPCRs), the role of the first intracellular loop (IL1) and its connections with adjacent transmembrane (TM) regions have not been investigated. Notably, these regions harbor several polar residues such as Ser and Thr. To begin uncovering how these polar residues may contribute to the structural basis for GPCR functionality, we have designed human D1-class receptor mutants (hD1-ST1 and hD5-ST1) whereby all Ser and Thr of IL1 and IL1/TM2 juncture have been replaced by Ala and Val, respectively. Both ST1 mutants exhibited a loss of dopamine affinity but similar binding properties for inverse agonists compared to their parent receptors. As well, these mutations diminished receptor activation for both subtypes, as indicated by an ablated constitutive activity and a pronounced decrease in dopamine potency. Interestingly, both mutants exhibited enhanced dopamine-mediated maximal stimulation (Emax) of adenylyl cyclase that was at least two-fold higher than wild-type. Point mutations for hD1R revealed that the loss in dopamine affinity and potency was attributed to Thr59, while the enhanced Emax of adenylyl cyclase was directly influenced by Ser65. These two residues are conserved among many Family A GPCRs and have recurring molecular interactions among crystallized structures. As such, their functional roles for IL1 and its transition into TM2 reported herein may also be applicable to other GPCRs. Our work thus potentially highlights a structural role of Thr59 and Ser65 in the formation of critical intramolecular interactions for ligand binding and signal transduction of D1-class dopaminergic receptors.

Asn441 plays a key role in folding and function of the Na + /I – symporter (NIS)

The Na(+)/I(-) symporter (NIS) is a plasma membrane glycoprotein that mediates active I(-) transport in the thyroid, the first step in the biosynthesis of the iodine-containing thyroid hormones T3 and T4. Several NIS mutants have been identified as a cause of congenital I(-) transport defect (ITD), and their investigation has yielded valuable mechanistic information on NIS. Here we report a thorough characterization of the ITD-causing NIS mutation in which the sixth intracellular loop residues 439-443 are missing. This mutant protein was intracellularly retained, incompletely glycosylated, and intrinsically inactive. Engineering 5 Ala at positions 439-443 partially recovered cell surface targeting and activity (∼15%). Strikingly, NIS with the sequence 439-AANAA-443, in which Asn was restored at position 441, was targeted to the plasma membrane and exhibited ∼95% the transport activity of WT NIS. Based on our NIS homology model, we propose that the side chain of N441, a residue conserved throughout most of the SLC5 family, interacts with the main chain amino group of G444, capping the α-helix of transmembrane segment XII and thus stabilizing the structure of the molecule. Our data provide insight into a critical interhelical interaction required for NIS folding and activity.

Abstract 897: Role of intracellular loops 1 and 3 in folding and cell surface expression of human P-glycoprotein (ABCB1).

Abstract P-glycoprotein (ABCB1) is an ATP-Binding Cassette (ABC) transporter that effluxes a variety of structurally diverse compounds including anticancer drugs out of the cell. Known crystal structures of ABC transporters show that the transmembrane domain (TMD) is connected to the nucleotide-binding domain (NBD) through a ball and socket joint. Also, the intracellular loops (ICLs) 1 and 3 are shown to be in close proximity to the NBDs, creating an extensive interaction surface between the TMDs and NBDs. Computational models of human P-gp in the apo and nucleotide-bound conformation have been developed based on structures of mouse mdr1a, C. elegans P-gp, Sav1866 and MsbA transporters. In these models, the adenine group of ATP forms hydrogen bonds with conserved D164 and D805 on ICLs 1 and 3, respectively, which are located at the interface between the NBDs and TMDs. We investigated the role of D164 and D805 by substituting these residues with cysteine in a cys-less background. The D164C/D805C mutant P-gp was expressed in High-Five insect cells as well as in HeLa cells using the BacMam baculovirus expression system. In the insect cells grown at 27°C, the expression of the mutant P-gp was comparable to that of cysless-WT protein and exhibited the same ATP-binding affinity but a lower Vmax for ATP hydrolysis. However, when the mutant protein was expressed in HeLa cells at 37°C, we found that the D164C/D805C P-gp was mislocalized and failed to transport almost all tested drug substrates. HeLa cells transduced with D164C/D805C mutant baculovirus were grown for 16-18 hrs in the presence of cyclosporine A (CsA), a substrate that is also known to function as a chemical chaperone. This treatment resulted in expression of mutant P-gp at the cell surface to the same extent as that of cysless-WT protein; similarly, the transport was also restored to the level of cysless-WT P-gp. A partial rescue of the mutant P-gp expression and function was observed in the presence of the CFTR correctors VRT-325 and compound-4a. In addition, when BacMam baculovirus-transduced HeLa cells were cultured at 27°C for 40 hrs, the mutant protein rescued to the cell surface with transport activity to the same extent as cysless-WT P-gp. Studies of each mutant form separately revealed that the D164C mutant had intermediate cell surface expression and was rescued completely to the plasma membrane in the presence of CsA. On the other hand, the expression of D805C at the cell surface was drastically decreased and even when HeLa cells were grown in the presence of CsA, only 10-15% of mutant protein was expressed at the cell surface compared to cysless-WT protein. These findings in aggregate illustrate the role of residues in ICLs 1 and 3 in the proper folding of P-gp and specifically show that the D164 and D805 residues are not critical for ATP binding or hydrolysis as proposed earlier, but are instead important for inter-domain interactions and assembly of a functional transporter. Citation Format: Khyati Kapoor, Jaya Bhatnagar, Eduardo E. Chufan, Suresh V. Ambudkar. Role of intracellular loops 1 and 3 in folding and cell surface expression of human P-glycoprotein (ABCB1). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 897. doi:10.1158/1538-7445.AM2013-897

Amino acids within intracellular loop 3 of the D2 dopamine receptor are necessary for arrestin binding and receptor internalization

Arrestins facilitate dopamine D2 receptor internalization and desensitization and are important mediators of dopaminergic behaviors. Using bioluminescence resonance energy transfer (BRET) to measure receptor‐mediated recruitment of arrestin, our objective was to determine the smallest mutation that prevents arrestin binding while retaining signaling through G‐proteins. Mutation of four residues in intracellular loop three (212–215, IYIV) abolished arrestin recruitment and internalization, but impaired receptor expression. Mutation of three (YIV) or two (IV) residues reduced arrestin recruitment by forty and thirty percent respectively, without impairing receptor expression. Importantly, mutating three residues rendered the receptor incapable of internalization, even when over‐expressing arrestin3 and GRK2, while sparing the receptor's ability to signal through G‐proteins. Thus, mutation of the sequence YIV in IC3 of the D2 receptor selectively impaired arrestin recruitment and abolished receptor internalization, suggesting that this mutant will be a useful tool for elucidating cellular and behavioral effects of D2 receptor stimulation that are mediated by arrestins. (VHA, MH045372 and DA007262)

Molecular Determinants in Glycine and GABA-A Receptors that Sense Intracellular Chloride Concentration

GABA-A and glycine receptors are ligand-gated ion channels belonging to the Cys-loop superfamily. They are permeable to anions and mediate fast synaptic inhibition in the central nervous system. We recently showed that intracellular chloride can affect the time course of deactivation of these channels (1). The aim of the present work was to identify the residues that are involved in “sensing” the intracellular chloride concentration.We used the fast concentration jump technique to apply brief pulses (1-3 ms) of saturating glycine or GABA to outside-out patches from transiently-transfected HEK cells expressing alpha1 or alpha1 beta glycine receptors or alpha1 beta2 gamma2L GABA-A receptors. The effect of intracellular chloride could be mediated either by residues in intracellular loops, such as M1-M2 and M3-M4, or by the residues lining the channel itself. We used a combination of deletion and Ala/Cys-scanning mutagenesis approaches to address this question.In other nicotinic channels, the long M3-M4 intracellular loop can be replaced by the equivalent domain (7 amino acids) of the orthologous prokaryotic channel from Gloeobacter violaceus without losing channel function (2). We found that chimeric constructs of glycine and GABA-A receptors containing the prokaryotic M3-M4 domain retained modulation by intracellular chloride, suggesting that the major cytoplasmic domain is unlikely to mediate this effect. The results of mutagenesis of residues facing the ion permeation pathway on the contrary strongly suggests that chloride ions affect channel kinetic by binding to a site along the pore; we have identified conserved residues that reduce the differences between the deactivation rates measured at different chloride concentrations.(1) Pitt, Sivilotti and Beato (2008) J.Neurosci 28, 11454-11467(2) Jansen, Bali and Akabas (2008) J.Gen.Physiol. 131, 137-146

Cytoplasmic Tail of Phospholemman Interacts with the Intracellular Loop of the Cardiac Na+/Ca2+Exchanger

Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na+/Ca2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the first five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using glutathione S-transferase (GST) pull-down assay, we demonstrated that the intracellular loop, but not the N- or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na+/Ca2+ exchangers consisting of N- or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. Although expression of N-terminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N- and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Co-immunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.

Functional Complementation and the Analysis of Opioid Receptor Homodimerization

Complementation of function after coexpression of pairs of nonfunctional G protein-coupled receptors that contain distinct inactivating mutations supports the hypothesis that such receptors exist as dimers. Chimeras between members of the metabotropic glutamate receptor-like family have been particularly useful because the N-terminal ligand binding and heptahelical transmembrane elements can be considered distinct domains. To examine the utility of a related approach for opioid receptors, fusion proteins were generated in which a pertussis toxin-resistant (Cys351Ile) variant of the G protein Gi1alpha was linked to the C-terminal tails of the delta opioid peptide (DOP), kappa opioid peptide, and mu opioid peptide receptors. Each was functional as measured by agonist stimulation of guanosine 5'-([gamma-35S]thio)triphosphate ([35S]GTPgammaS) binding in Gialpha immunoprecipitates from membranes of pertussis toxin-treated HEK293 cells. Agonist function was eliminated either by fusion of the receptors to Gi1alphaGly202Ala,Cys351Ile or mutation of a pair of conserved Val residues in intracellular loop 2 of each receptor. Coexpression, but not simple mixing, of the two inactive fusion proteins reconstituted agonist-loading of [35S]GTPgammaS for each receptor. At equimolar amounts, reconstitution of DOP receptor function was more extensive than kappa or mu opioid receptor. Reconstitution of DOP function required two intact receptors and was not achieved by provision of extra Gi1alphaCys351Ile membrane anchored by linkage to DOP transmembrane domain 1. Inactive forms of all G protein alpha subunits can be produced by mutations equivalent to Gi1alphaGly202Ala. Because the amino acids modified in the opioid receptors are highly conserved in most rhodopsin-like receptors, this approach should be widely applicable to study the existence and molecular basis of receptor dimerization.

The association of arrestin-3 with the follitropin receptor depends on receptor activation and phosphorylation

We have recently shown that the binding of arrestin-3 to the lutropin receptor (LHR) is dependent mostly on receptor activation rather than on phosphorylation. The experiments presented here were designed to test the involvement of these two events in the association of arrestin-3 with the closely related follitropin receptor (FSHR). Activation of the FSHR leads to the phosphorylation of residues in the first and third intracellular loops. Mutation of the phosphorylation sites in the third intracellular loop of the rat (r) FSHR partially reduces phosphorylation but has no effect on arrestin-3 association. Mutation of the phosphorylation sites in the first intracellular loop abolishes phosphorylation and arrestin-3 association. Dominant-negative mutants of G protein-coupled receptor kinase (GRKs) 2 and 6 inhibit rFSHR phosphorylation to the same extent but only the dominant-negative mutant of GRK2 inhibits arrestin-3 association. Two mutations of the rFSHR (D389N and Y530F) that impair activation and abolish phosphorylation also impair arrestin-3 binding. GRK2 restores the phosphorylation of both mutants but it restores arrestin-3 association only to the D389N mutant. We conclude that, in contrast to the data obtained with the LHR, the association of arrestin-3 with the FSHR is dependent on receptor phosphorylation. The phosphorylation of the third intracellular loop residues is not needed for arrestin-3 association, however.

Multiple domains interacting with Gs in the porcine calcitonin receptor.

The molecular basis for Gs activation by the calcitonin (CT) receptor was investigated. Based upon the analysis of conserved regions in G protein-coupled receptors, two nonoverlapping regions in the heptahelical porcine CT receptor (CTR) were selected as candidate Gs-interacting domains: the third intracellular loop residues 327-344 (KLKESQEAESHMYLKAVR, P3 region) and the C-tail residues 404-418 (KRQWNQYQAQRWAGR, P4 region). To assess their Gs-interacting function, we expressed these sequences in hybrid insulin-like growth factor II receptors in which the receptor native Gi-interacting domain was converted to CTR sequences. In COS cells transfected with either P3- or P4-substituted hybrid receptor, membrane adenylyl cyclase activity significantly increased. The up-regulated activity of cAMP was confirmed by measuring the transcriptional activity of the cAMP response element in cells expressing either hybrid receptor. A mutant CTR lacking the P4 region maintained positive cAMP response but with an attenuated maximal capacity to produce cAMP. In contrast, we could not assess the function of the P3 region using a conventional deletion method, as CT bound poorly to cells transfected with either of the two P3-deficient CTRs (one lacking the P3 region and the other lacking P3 but having the P3 sequence in reverse orientation). These data suggest that the third intracellular loop and the C-tail in CTR have domain-specific roles in Gs activation and that the hybrid receptor approach used here, combined with a conventional mutagenesis approach, is useful for intact cell analysis and functional dissection of G protein-coupled receptors.

Evidence for an important functional role of intracellular loop II of the lutropin receptor

The lutropin receptor (LHR) is a G protein-coupled receptor in which high affinity ligand binding occurs to the relatively large extracellular N-terminal domain. Various portions of the receptor have been mapped for their relative importance in hormone-mediated signaling. There is, however, a paucity of information available on the intracellular loops (ICL), where, along with the C-terminal cytoplasmic tail, G protein coupling is expected to occur. Site-directed mutagenesis was used to investigate the role of several conserved ionizable groups and one tyrosyl residue in ICLs I–III of the rat LHR. The pSVL expression vector, containing the LHR cDNA (wild-type and mutants), was transiently transfected into COS-7 cells, and human choriogonadotropin (hCG) binding and hCG-mediated cAMP production were determined. Several point mutants of amino acid residues in ICL II were prepared and characterized with the following results: replacements of Lys-455 and of His-460 with Glu gave mutant LHRs that failed to localize or fold properly at the cell surface as evidenced by the lack of significant binding to intact cells, although hCG binding could be detected in broken cell preparations, and a neighboring Arg-459→Glu replacement had no apparent effect on receptor trafficking, hCG binding or hCG-mediated cAMP-production. A reversal mutant in ICL II in which Glu-441, at the boundary of transmembrane helix III and ICL II, and His-460, at the interface between ICL II and transmembrane helix IV, were interchanged, exhibited hCG binding to intact cells, but the maximal cAMP level at high concentrations of ligand was less than that obtained with COS-7 cells transfected with wild-type LHR. The total number of cell surface receptors determined with the reversal mutant was less than that found with wild-type LHR. This difference, however, is not believed to be responsible for the reduced signaling, since maximal cAMP responses to hCG were obtained with comparable receptor densities of wild-type and various mutant LHRs. Other single replacements in ICL I, Lys-368→Glu and to Gln, and in ICL III, Arg-526→Glu and Tyr-528→Ser, resulted in mutant LHRs with characteristics of wild-type LHR in trafficking, hCG binding and hCG-mediated cAMP production. These findings suggest an important functional role of several amino acid residues in ICL II of LHR.