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Insertion of isolated insulin receptors into placental membrane vesicles.

ABSTRACT: Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid.

SUBMITTER: Christiansen K

PROVIDER: S-EPMC1130702 | biostudies-other | 1992 Jan

REPOSITORIES: biostudies-other

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Project description:Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid composition of membranes. Here, using a newly developed chemical engineering procedure, we report on a bead-supported unilamellar membrane (bSUM) system that allows good control over membrane dimension, protein orientation, and lipid composition. Our new system uses specific ligands to facilitate the unidirectional incorporation of membrane proteins into lipid bilayers. Cryo-electron microscopic imaging demonstrates the unilamellar nature of the bSUMs. Electrical recordings from voltage-gated ion channels in bSUMs of varying diameters demonstrate the versatility of the new system. Using KvAP as a model system, we show that compared with other in vitro membrane systems, the bSUMs have the following advantages: (a) a major fraction of channels are orientated in a controlled way; (b) the channels mediate the formation of the lipid bilayer; (c) there is one and only one bilayer membrane on each bead; (d) the lipid composition can be controlled and the bSUM size is also under experimental control over a range of 0.2-20 µm; (e) the channel activity can be recorded by patch clamp using a planar electrode; and (f) the voltage-clamp speed (0.2-0.5 ms) of the bSUM on a planar electrode is fast, making it suitable to study ion channels with fast gating kinetics. Our observations suggest that the chemically engineered bSUMs afford a novel platform for studying lipid-protein interactions in membranes of varying lipid composition and may be useful for other applications, such as targeted delivery and single-molecule imaging.

Project description:BackgroundDiabetes, obesity, and overweight are prevalent pregnancy complications that predispose offspring to neurodevelopmental disorders, including autism, attention-deficit/hyperactivity disorder, and schizophrenia. Although male individuals are three to four times more likely than female individuals to develop these disorders, the mechanisms driving the sex specificity of disease vulnerability remain unclear. Because defective placental insulin receptor (InsR) signaling is a hallmark of pregnancy metabolic dysfunction, we hypothesized that it may be an important contributor and novel mechanistic link to sex-specific neurodevelopmental changes underlying disease risk.MethodsWe used Cre/loxP transgenic mice to conditionally target InsRs in fetally derived placental trophoblasts. Adult offspring were evaluated for effects of placental trophoblast-specific InsR deficiency on stress sensitivity, cognitive function, sensorimotor gating, and prefrontal cortical transcriptional reprogramming. To evaluate molecular mechanisms driving sex-specific outcomes, we assessed genome-wide expression profiles in the placenta and fetal brain.ResultsMale, but not female, mice with placental trophoblast-specific InsR deficiency showed a significantly increased hypothalamic-pituitary-adrenal axis stress response and impaired sensorimotor gating, phenotypic effects that were associated with dysregulated nucleotide metabolic processes in the male prefrontal cortex. Within the placenta, InsR deficiency elicited changes in gene expression, predominantly in male mice, reflecting potential shifts in vasculature, amino acid transport, serotonin homeostasis, and mitochondrial function. These placental disruptions were associated with altered gene expression profiles in the male fetal brain and suggested delayed cortical development.ConclusionsTogether, these data demonstrate the novel role of placental InsRs in sex-specific neurodevelopment and reveal a potential mechanism for neurodevelopmental disorder risk in pregnancies complicated by maternal metabolic disorders, including diabetes and obesity.

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Insertion of isolated insulin receptors into placental membrane vesicles.

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