Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein

1985 ◽  
Vol 5 (6) ◽  
pp. 1442-1448
Author(s):  
G A Adams ◽  
J K Rose
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
G Protein ◽  
Abnormal Behavior ◽  
Animal Cells ◽  
Viral Glycoprotein ◽  
Protein Levels ◽  
Membrane Spanning ◽  
Membrane Anchoring ◽  
Isoleucine Residue

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G protein) consists of a continuous stretch of 20 uncharged and mostly hydrophobic amino acids. We examined the effects of two mutations which change the amino acid sequence in this domain. These mutations were generated by oligonucleotide-directed mutagenesis of a cDNA clone encoding the G protein, and the altered G proteins were then expressed in animal cells. Replacement of an isoleucine residue in the center of this domain with a strongly polar but uncharged amino acid (glutamine) had no effect on membrane anchoring or transport of the protein to the cell surface. Replacement of this same isoleucine residue with a charged amino acid (arginine) generated a G protein that still spanned intracellular membranes but was not transported efficiently to the cell surface. The protein accumulated in the Golgi region in about 50% of the cells, and about 20% of the cells had detectable protein levels in a punctate pattern on the cell surface. In the remaining cells the protein accumulated in a vesicular pattern throughout the cytoplasm. Models which might explain the abnormal behavior of this protein are discussed.

scholarly journals Incorporation of a charged amino acid into the membrane-spanning domain blocks cell surface transport but not membrane anchoring of a viral glycoprotein.

1985 ◽  
Vol 5 (6) ◽  
pp. 1442-1448 ◽  
Author(s):  
G A Adams ◽  
J K Rose
Keyword(s):  
Amino Acid ◽  
Cell Surface ◽  
G Protein ◽  
Abnormal Behavior ◽  
Animal Cells ◽  
Viral Glycoprotein ◽  
Protein Levels ◽  
Membrane Spanning ◽  
Membrane Anchoring ◽  
Isoleucine Residue

The membrane-spanning domain of the vesicular stomatitis virus glycoprotein (G protein) consists of a continuous stretch of 20 uncharged and mostly hydrophobic amino acids. We examined the effects of two mutations which change the amino acid sequence in this domain. These mutations were generated by oligonucleotide-directed mutagenesis of a cDNA clone encoding the G protein, and the altered G proteins were then expressed in animal cells. Replacement of an isoleucine residue in the center of this domain with a strongly polar but uncharged amino acid (glutamine) had no effect on membrane anchoring or transport of the protein to the cell surface. Replacement of this same isoleucine residue with a charged amino acid (arginine) generated a G protein that still spanned intracellular membranes but was not transported efficiently to the cell surface. The protein accumulated in the Golgi region in about 50% of the cells, and about 20% of the cells had detectable protein levels in a punctate pattern on the cell surface. In the remaining cells the protein accumulated in a vesicular pattern throughout the cytoplasm. Models which might explain the abnormal behavior of this protein are discussed.

scholarly journals Role of nutrient-sensing taste 1 receptor (T1R) family members in gastrointestinal chemosensing

2014 ◽  
Vol 111 (S1) ◽  
pp. S8-S15 ◽  
Author(s):  
Soraya P. Shirazi-Beechey ◽  
Kristian Daly ◽  
Miran Al-Rammahi ◽  
Andrew W. Moran ◽  
David Bravo
Keyword(s):  
Amino Acid ◽  
G Protein ◽  
Gut Hormones ◽  
Glucose Absorption ◽  
Nutrient Sensing ◽  
Acid Activation ◽  
Protein Levels ◽  
Apical Domain ◽  
The Impact

Luminal nutrient sensing by G-protein-coupled receptors (GPCR) expressed on the apical domain of enteroendocrine cells activates intracellular pathways leading to secretion of gut hormones that control vital physiological processes such as digestion, absorption, food intake and glucose homeostasis. The taste 1 receptor (T1R) family of GPCR consists of three members: T1R1; T1R2; T1R3. Expression of T1R1, T1R2 and T1R3 at mRNA and protein levels has been demonstrated in the intestinal tissue of various species. It has been shown that T1R2–T1R3, in association with G-protein gustducin, is expressed in intestinal K and L endocrine cells, where it acts as the intestinal glucose (sweet) sensor. A number of studies have demonstrated that activation of T1R2–T1R3 by natural sugars and artificial sweeteners leads to secretion of glucagon-like peptides 1&2 (GLP-1 and GLP-2) and glucose dependent insulinotropic peptide (GIP). GLP-1 and GIP enhance insulin secretion; GLP-2 increases intestinal growth and glucose absorption. T1R1–T1R3 combination co-expressed on the apical domain of cholecystokinin (CCK) expressing cells is a luminal sensor for a number of l-amino acids; with amino acid-activation of the receptor eliciting CCK secretion. This article focuses on the role of the gut-expressed T1R1, T1R2 and T1R3 in intestinal sweet and l-amino acid sensing. The impact of exploiting T1R2–T1R3 as a nutritional target for enhancing intestinal glucose absorption and gut structural maturity in young animals is also highlighted.

scholarly journals Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: toward establishing rules governing the lateral mobility of membrane proteins.

1991 ◽  
Vol 115 (1) ◽  
pp. 75-84 ◽  
Author(s):  
F Zhang ◽  
B Crise ◽  
B Su ◽  
Y Hou ◽  
J K Rose ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Cell Surface ◽  
High Mobility ◽  
Lateral Diffusion ◽  
Extracellular Domain ◽  
Major Determinant ◽  
Animal Cells ◽  
Lateral Mobility ◽  
Vesicular Stomatitis ◽  
Membrane Spanning

In the plasma membrane of animal cells, many membrane-spanning proteins exhibit lower lateral mobilities than glycosylphosphatidylinositol (GPI)-linked proteins. To determine if the GPI linkage was a major determinant of the high lateral mobility of these proteins, we measured the lateral diffusion of chimeric membrane proteins composed of normally transmembrane proteins that were converted to GPI-linked proteins, or GPI-linked proteins that were converted to membrane-spanning proteins. These studies indicate that GPI linkage contributes only marginally (approximately twofold) to the higher mobility of several GPI-linked proteins. The major determinant of the high mobility of these proteins resides instead in the extracellular domain. We propose that lack of interaction of the extracellular domain of this protein class with other cell surface components allows diffusion that is constrained only by the diffusion of the membrane anchor. In contrast, cell surface interactions of the ectodomain of membrane-spanning proteins exemplified by the vesicular stomatitis virus G glycoprotein reduces their lateral diffusion coefficients by nearly 10-fold with respect to many GPI-linked proteins.

Surface Structure of Nucleated Animal Cells: Carbon Replicas

1967 ◽  
Vol 25 ◽  
pp. 120-121
Author(s):  
Robert M. Glaeser ◽  
Thea B. Scott
Keyword(s):  
Cell Surface ◽  
Surface Structure ◽  
Particle Diameter ◽  
Cellular Functions ◽  
Animal Cells ◽  
Replica Technique ◽  
Carbon Replica ◽  
Characteristic Particle ◽  
Cell Surface Structure ◽  
Carbon Replicas

The carbon-replica technique can be used to obtain information about cell-surface structure that cannot ordinarily be obtained by thin-section techniques. Mammalian erythrocytes have been studied by the replica technique and they appear to be characterized by a pebbly or “plaqued“ surface texture. The characteristic “particle” diameter is about 200 Å to 400 Å. We have now extended our observations on cell-surface structure to chicken and frog erythrocytes, which possess a broad range of cellular functions, and to normal rat lymphocytes and mouse ascites tumor cells, which are capable of cell division. In these experiments fresh cells were washed in Eagle's Minimum Essential Medium Salt Solution (for suspension cultures) and one volume of a 10% cell suspension was added to one volume of 2% OsO4 or 5% gluteraldehyde in 0.067 M phosphate buffer, pH 7.3. Carbon replicas were obtained by a technique similar to that employed by Glaeser et al. Figure 1 shows an electron micrograph of a carbon replica made from a chicken erythrocyte, and Figure 2 shows an enlarged portion of the same cell.

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