Lipid-protein interactions in GPCR-associated signaling

Signal transduction via G-protein-coupled receptors (GPCRs) is a fundamental pathway through which the functions of an individual cell can be integrated within the demands of a multicellular organism. Since this family of receptors first discovered, the proteins that constitute this signaling cascade and their interactions with one another have been studied intensely. In parallel, the pivotal role of lipids in the correct and efficient propagation of extracellular signals has attracted ever increasing attention. This is not surprising given that most of the signal transduction machinery is membrane-associated and therefore lipid-related. Hence, lipid-protein interactions exert a considerable influence on the activity of these proteins. This review focuses on the post-translational lipid modifications of GPCRs and G proteins (palmitoylation, myristoylation, and isoprenylation) and their significance for membrane binding, trafficking and signaling. Moreover, we address how the particular biophysical properties of different membrane structures may regulate the localization of these proteins and the potential functional consequences of this phenomenon in signal transduction. Finally, the interactions that occur between membrane lipids and GPCR effector enzymes such as PLC and PKC are also considered.     

Lipid-protein interactions in GPCR-associated signaling

Signal transduction via G-protein-coupled receptors (GPCRs) is a fundamental pathway through which the functions of an individual cell can be integrated within the demands of a multicellular organism. Since this family of receptors first discovered, the proteins that constitute this signaling cascade and their interactions with one another have been studied intensely. In parallel, the pivotal role of lipids in the correct and efficient propagation of extracellular signals has attracted ever increasing attention. This is not surprising given that most of the signal transduction machinery is membrane-associated and therefore lipid-related. Hence, lipid-protein interactions exert a considerable influence on the activity of these proteins. This review focuses on the post-translational lipid modifications of GPCRs and G proteins (palmitoylation, myristoylation, and isoprenylation) and their significance for membrane binding, trafficking and signaling. Moreover, we address how the particular biophysical properties of different membrane structures may regulate the localization of these proteins and the potential functional consequences of this phenomenon in signal transduction. Finally, the interactions that occur between membrane lipids and GPCR effector enzymes such as PLC and PKC are also considered.     

High expression of growth factor receptor-bound protein 14 predicts poor prognosis for colorectal cancer patients

Objects

To explore the roles of growth factor receptor-bound protein 14 (GRB14) in colorectal cancer (CRC) and its correlation with clinicopathological characteristics and prognosis of CRC patients.

Results

GRB14 was localized in the cytoplasm of CRC and benign glandular epithelium cells, showing higher levels in CRC tissues compared with normal colon samples (P < 0.001). High GRB14 was associated with a high pathological grade (P = 0.045), advanced clinical stage (P = 0.018), enhanced tumor invasion (P < 0.001) and lymph node metastasis (P = 0.028). The cancer genome atlas (TCGA) mRNA sequence data showed that GRB14 was upregulated in CRC at an advanced clinical stage (P = 0.011) with enhanced tumor invasion (P < 0.001) and lymph node metastasis (P = 0.014). Kaplan–Meier survival curves revealed that CRC patients with high GRB14 levels had a shorter survival compared with those showing low GRB14 expression (P = 0.007). High GRB14 expression was an independent prognostic factor for CRC patients (HR 2.847, 95 %CI 1.058–7.659; P = 0.038).

Conclusions

GRB14 may be an important cancer promoter that enhances CRC progression. Upregulated GRB14 levels may predict a poor clinical outcome in CRC patients.

     

Insulin-like growth factor 1/insulin signaling activates androgen signaling through direct interactions of Foxo1 with androgen receptor

The androgen-androgen receptor (AR) system plays vital roles in a wide array of biological processes, including prostate cancer development and progression. Several growth factors, such as insulin-like growth factor 1 (IGF1), can induce AR activation, whereas insulin resistance and hyperinsulinemia are correlated with an elevated incidence of prostate cancer. Here we report that Foxo1, a downstream molecule that becomes phosphorylated and inactivated by phosphatidylinositol 3-kinase/Akt kinase in response to IGF1 or insulin, suppresses ligand-mediated AR transactivation. Foxo1 reduces androgen-induced AR target gene expressions and suppresses the in vitro growth of prostate cancer cells. These inhibitory effects of Foxo1 are attenuated by IGF1 but are enhanced when it is rendered Akt-nonphosphorylatable. Foxo1 interacts directly with the C terminus of AR in a ligand-dependent manner and disrupts ligand-induced AR subnuclear compartmentalization. Foxo1 is recruited by liganded AR to the chromatin of AR target gene promoters, where it interferes with AR-DNA interactions. IGF1 or insulin abolish the Foxo1 occupancy of these promoters. Of interest, a positive feedback circuit working locally in an autocrine/intracrine manner may exist, because liganded AR up-regulates IGF1 receptor expression in prostate cancer cells, presumably resulting in higher IGF1 signaling tension and further enhancing the functions of the receptor itself. Thus, Foxo1 is a novel corepressor for AR, and IGF1/insulin signaling may confer stimulatory effects on AR by attenuating Foxo1 inhibition. These results highlight the potential involvement of metabolic syndrome and hyperinsulinemia in prostate diseases and further suggest that intervention of IGF1/insulin-phosphatidylinositol 3-kinase-Akt signaling may be of clinical value for prostate diseases.     

Lipid-protein interactions in reconstituted membranes containing acetylcholine receptor

Functional membranes containing purified Torpedo californica acetylcholine receptor and dioleoylphosphatidylcholine (DOPC) were prepared by a cholate dialysis procedure with lipid to protein ratios of 100-400 to 1 (mol/mol). Spin-labeled lipids were incorporated into the reconstituted membranes and into native membranes prepared from Torpedo electroplax, and electron paramagnetic resonance (EPR) spectra were recorded between 0 and 20 degrees C. The spin-labels included nitroxide derivatives of stearic acid (16-doxylstearic acid), androstane, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA). The phospholipid spin-labels had 16-doxylstearic acid in the sn-2 position. All the spectra showed two components corresponding to a relatively mobile bilayer component and a motionally restricted "protein-perturbed" component. The relative amounts of mobile and perturbed components were quantitated by spectral subtraction and integration techniques. The mobile/perturbed ratio was somewhat temperature dependent, and the results are discussed in terms of exchange between mobile and perturbed environments. Plots of the mobile/perturbed ratios vs. lipid/protein ratios at 1 degree C gave straight lines from which the relative binding affinity of each spin-label and the number of perturbed lipids per receptor protein could be calculated. All the spin-labels gave similar values for the number of perturbed lipids (40 +/- 7), a number close to the number of lipids that will fit around the intramembranous perimeter of the receptor. The affinities of the spin-labeled lipids for the receptor relative to DOPC were androstane (K = 4.3) congruent to 16-doxylstearic acid (4.1) greater than PA (2.7) greater than PE (1.1) approximately PC (1.0) approximately PS (0.7). The lipids having the highest affinity for the acetylcholine receptor were also those that have the largest effects on the ion flux functional properties of the receptor, and the results are discussed in terms of lipid effects on receptor function.     

Growth factor receptor-bound protein 14: a new modulator of photoreceptor-specific cyclic-nucleotide-gated channel

Growth factor receptor-bound protein 14 (Grb14) is an adaptor protein that is involved in receptor tyrosine kinase signalling. In this study, we report that Grb14 interacts with the rod photoreceptor-specific cyclic-nucleotide-gated channel alpha subunit (CNGA1) and decreases its affinity for cyclic guanosine monophosphate. Channel modulation is controlled by direct binding of the Grb14 Ras-associating domain with the carboxy-terminal region of CNGA1. We observed that the channel remains open in Grb14(-/-) mice that are exposed to light, suggesting that Grb14 is a normal physiological modulator of CNG channel function in vivo.     

Diversification of Neu differentiation factor and epidermal growth factor signaling by combinatorial receptor interactions.

The ErbB family includes two receptors, ErbB-1 and ErbB-3, that respectively bind to epidermal growth factor and Neu differentiation factor, and an orphan receptor, ErbB-2. Unlike ErbB-1 and ErbB-2, the intrinsic tyrosine kinase of ErbB-3 is catalytically impaired. By using interleukin-3-dependent cells that ectopically express the three ErbB proteins or their combinations, we found that ErbB-3 is devoid of any biological activity but both ErbB-1 and ErbB-2 can reconstitute its extremely potent mitogenic activity. Transactivation of ErbB-3 correlates with heterodimer formation and is reflected in receptor phosphorylation and the transregulation of ligand affinity. Inter-receptor interactions enable graded proliferative and survival signals: heterodimers are more potent than homodimers, and ErbB-3-containing complexes, especially the ErbB-2/ErbB-3 heterodimer, are more active than ErbB-1 complexes. Nevertheless, ErbB-1 signaling displays dominance over ErbB-3 when the two receptors are coexpressed. Although all receptor combinations activate the mitogen-activated protein kinases ERK and c-Jun kinase, they differ in their rate of endocytosis and in coupling to intervening signaling proteins. It is conceivable that combinatorial receptor interactions diversify signal transduction and confer double regulation, in cis and in trans, of the superior mitogenic activity of the kinase-defective ErbB-3.     

Lipid-protein interactions in lipovitellin

The refined molecular structure of lipovitellin is described using synchrotron cryocrystallographic data to 1.9 A resolution. Lipovitellin is the predominant lipoprotein found in the yolk of egg-laying animals and is involved in lipid and metal storage. It is thought to be related in amino acid sequence to segments of apolipoprotein B and the microsomal transfer protein responsible for the assembly of low-density lipoproteins. Lipovitellin contains a heterogeneous mixture of about 16% (w/w) noncovalently bound lipid, mostly phospholipid. Previous X-ray structural studies at ambient temperature described several different protein domains including a large cavity in each subunit of the dimeric protein. The cavity was free of any visible electron density for lipid molecules at room temperature, suggesting that only dynamic interactions exist with the protein. An important result from this crystallographic study at 100 K is the appearance of some bound ordered lipid along the walls of the binding cavity. The precise identification of the lipid type is difficult because of discontinuities in the electron density. Nonetheless, the conformations of 7 phospholipids and 43 segments of hydrocarbon chains greater than 5 atoms in length have been discovered. The conformations of the bound lipid and the interactions between protein and lipid provide insights into the factors governing lipoprotein formation.     

Lipid-protein interactions in membranes

The interactions of lipids with integral and peripheral proteins can be studied in reconstituted and natural membranes using spin label electron spin resonance (ESR) spectroscopy. The ESR spectra reveal a reduction in mobility of the spin-labelled lipid species, and in certain cases evidence is obtained for a partial penetration of the peripheral proteins into the membrane. The latter may be relevant to the import mechanism of apocytochrome c into mitochondria. Integral proteins induce a more direct motional restriction of the spin-labelled lipid chains, allowing the stoichiometry and specificity of the interaction, and the lipid exchange rate at the protein interface to be determined from the ESR spectra. In this way, a population of very slowly exchanging cardiolipin associated with the mitochondrial ADP-ATP carrier has been identified. The residues involved in the specificity for charged lipids of the myelin proteolipid protein have been localized to the deletion in the DM-20 mutant, and the difference in lipid-protein interactions with the beta-sheet and alpha-helical conformations of the M-13 coat protein, has been characterized.     

Grb10 adapter protein as regulator of insulin-like growth factor receptor signaling

Grb10 is a member of a superfamily of adapter proteins that includes Grb10, 7, 14, and a protein of Caenorhabditis elegans called Mig10. Grb10 proteins are binding partners for several trans-membrane tyrosine-kinase receptors, including the insulin-like growth factor receptor (IGF-IR) and the insulin receptor (IR). Many recent reports have suggested a very important role of Grb10 in regulating IGF-IR signaling. In this review, we will focus on the role of Grb10 in IGF-I-induced mitogenesis and we will discuss the recent findings that show the involvement of Grb10 in the regulation of ligand-induced ubiquitination, internalization, and stability of the IGF-IR.     

Affinity chromatography for purification of the modular protein growth factor receptor-bound protein 2 and development of a screening test for growth factor receptor-bound protein 2 Src homology 3 domain inhibitor using peroxidase-linked ligand

Growth factor receptor-bound protein 2 (Grb2) is an adapter protein involved in the Ras-dependent signaling pathway that plays an important role in human cancers initiated by oncogenic receptors. Grb2 is constituted by one Src homology 2 domain surrounded by two SH3 domains, and the inhibition of the interactions produced by these domains could provide an antitumor approach. In evaluating chemical libraries, to search for potential Grb2 inhibitors, it was necessary to elaborate a rapid test for their screening. We have developed, first, a batch method based on the use of an affinity column bearing a Grb2-SH3 peptide ligand to isolate highly purified Grb2. We subsequently describe a very rapid 96-well screening of inhibitors based on a simple competition between purified Grb2 and a peroxidase-coupled proline-rich peptide.     

Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases

Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites.     

Disruption of the insulin-like growth factor type 1 receptor in osteoblasts enhances insulin signaling and action

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulin-like growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (DeltaIGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In DeltaIGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, DeltaIGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-(3)H]-deoxy-d-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of DeltaIGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.     

Essential role of insulin and insulin-like growth factor 1 receptor signaling in cardiac development and function

Cardiovascular disease is the leading cause of death in people with type 2 diabetes and is linked to insulin resistance even in the absence of diabetes. Here we show that mice with combined deficiency of the insulin receptor and insulin-like growth factor 1 (IGF-1) receptor in cardiac and skeletal muscle develop early-onset dilated cardiomyopathy and die from heart failure within the first month of life despite having a normal glucose homeostasis. Mice lacking the insulin receptor show impaired cardiac performance at 6 months, and mice lacking the insulin receptor plus one Igf1r allele have slightly increased mortality. By contrast, mice lacking the IGF-1 receptor or the IGF-1 receptor plus one Ir allele appear normal. Morphological characterization and oligonucleotide array analysis of gene expression demonstrate that prior to development of these physiological defects, mice with combined deficiency of both insulin and IGF-1 receptors have a coordinated down-regulation of genes encoding components of the electron transport chain and mitochondrial fatty acid beta-oxidation pathways and altered expression of contractile proteins. Thus, while neither the insulin receptor nor IGF-1 receptor in muscle is critical for glucose homeostasis during the first month of life, signaling from these receptors, particularly the insulin receptor, is required for normal cardiac metabolism and function.     

GIPC participates in G protein signaling downstream of insulin-like growth factor 1 receptor.

Several recent studies have demonstrated that insulin-like growth factor (IGF)-1-induced mitogen-activated protein kinase (MAP kinase) activation is abolished by pertussis toxin, suggesting that trimeric G proteins of the G(i) class are novel cellular targets of the IGF-1 signaling pathway. We report here that the intracellular domain of the Xenopus IGF-1 receptor is capable of binding to the Xenopus homolog of mammalian GIPC, a PDZ domain-containing protein previously identified as a binding partner of G(i)-specific GAP (RGS-GAIP). Binding of xGIPC to xIGF-1 receptor is independent of the kinase activity of the receptor and appears to require the PDZ domain of xGIPC. Injection of two C-terminal truncation mutants that retained the PDZ domain blocked IGF-1-induced Xenopus MAP kinase activation and oocyte maturation. While full-length xGIPC injection did not significantly alter insulin response, it greatly enhanced human RGS-GAIP in stimulating the insulin response in frog oocytes. This represents the first demonstration that GIPC x RGS-GAIP complex acts positively in IGF-1 receptor signal transduction.