scholarly journals Persistent activation of the α subunit of Gs promotes its removal from the plasma membrane

1989 ◽  
Vol 260 (3) ◽  
pp. 837-841 ◽  
Author(s):  
G Milligan ◽  
C G Unson
Keyword(s):  
Plasma Membrane ◽  
Membrane Fraction ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Α Subunit ◽  
Rat Glioma ◽  
Glioma C6 ◽  
Membrane Attachment ◽  
Guanine Nucleotide Binding ◽  
Gs Alpha

As assessed both by cholera-toxin-catalysed ADP-ribosylation and by immunoblotting with an anti-peptide antiserum raised against the C-terminal decapeptide of forms of Gs alpha (the alpha subunit of the stimulatory guanine nucleotide-binding protein), rat glioma C6 BU1 cells express two forms of Gs alpha: a major 44 kDa form and a much less prevalent 42 kDa form. We examined the effects of guanine nucleotides on the interaction of the 44 kDa form with the plasma membrane. Incubation of membranes of C6 BU1 cells with poorly hydrolysed analogues of GTP, but not with analogues of either ATP or GDP, caused the release of this Gs alpha from the membrane fraction. Release of Gs alpha was observed within 5 min, and continued throughout the incubation period. After treatment with guanosine 5′-[beta gamma-imido]triphosphate for 60 min, some 75% of this polypeptide had been released from its site of membrane attachment. These experiments demonstrate that Gs alpha need not remain associated invariantly with the plasma membrane.

scholarly journals GTP analogues promote release of the α subunit of the guanine nucleotide binding protein, Gi2, from membranes of rat glioma C6 BU1 cells

1988 ◽  
Vol 254 (2) ◽  
pp. 391-396 ◽  
Author(s):  
G Milligan ◽  
I Mullaney ◽  
C G Unson ◽  
L Marshall ◽  
A M Spiegel ◽  
...  
Keyword(s):  
G Protein ◽  
Pertussis Toxin ◽  
Nucleotide Binding ◽  
Alpha Subunit ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Rat Glioma ◽  
Glioma C6 ◽  
Gamma Subunits ◽  
Guanine Nucleotide Binding

The major pertussis-toxin-sensitive guanine nucleotide-binding protein of rat glioma C6 BU1 cells corresponded immunologically to Gi2. Antibodies which recognize the alpha subunit of this protein indicated that it has an apparent molecular mass of 40 kDa and a pI of 5.7. Incubation of membranes of these cells with guanosine 5′-[beta gamma-imido]triphosphate, or other analogues of GTP, caused release of this polypeptide from the membrane in a time-dependent manner. Analogues of GDP or of ATP did not mimic this effect. The GTP analogues similarly caused release of the alpha subunit of Gi2 from membranes of C6 cells in which this G-protein had been inactivated by pretreatment with pertussis toxin. The beta subunit was not released from the membrane under any of these conditions, indicating that the release process was a specific response to the dissociation of the G-protein after binding of the GTP analogue. Similar nucleotide profiles for release of the alpha subunits of forms of Gi were noted for membranes of both the neuroblastoma x glioma hybrid cell line NG108-15 and of human platelets. These data provide evidence that: (1) pertussis-toxin-sensitive G-proteins, in native membranes, do indeed dissociate into alpha and beta gamma subunits upon activation; (2) the alpha subunit of ‘Gi-like’ proteins need not always remain in intimate association with the plasma membrane; and (3) the alpha subunit of Gi2 can still dissociate from the beta/gamma subunits after pertussis-toxin-catalysed ADP-ribosylation.

scholarly journals Foetal-calf serum stimulates a pertussis-toxin-sensitive high-affinity GTPase activity in rat glioma C6 BU1 cells

1987 ◽  
Vol 245 (2) ◽  
pp. 501-505 ◽  
Author(s):  
G Milligan
Keyword(s):  
Pertussis Toxin ◽  
Cellular Proliferation ◽  
Calf Serum ◽  
Foetal Calf Serum ◽  
Gtpase Activity ◽  
Guanine Nucleotide Binding Protein ◽  
Rat Glioma ◽  
High Affinity ◽  
Glioma C6 ◽  
Guanine Nucleotide Binding

Cellular proliferation of rat glioma C6 BU1 cells in tissue culture is dependent on the presence of either calf or foetal-calf serum in the medium. Foetal-calf serum stimulated a high-affinity GTPase in membranes derived from C6 BU1 cells. Pretreatment of the cells with pertussis toxin decreased the high-affinity GTPase activity substantially, and attenuated the foetal-calf-serum-stimulated increase in this GTPase activity. Cholera toxin, in contrast, did not modulate the response to foetal-calf serum. Foetal-calf serum did not inhibit adenylate cyclase activity in membranes of these cells, indicating that the G-protein that was stimulated by foetal-calf serum was not Gi (the inhibitory one). Although the nature of the specific component of foetal-calf serum responsible for this pertussis-toxin-sensitive receptor-mediated stimulation of high-affinity GTPase activity has not been identified, it was mimicked neither by bombesin, which can stimulate inositol phospholipid turnover via a guanine nucleotide binding protein, nor by platelet-derived growth factor, which is present in substantial concentrations in foetal-calf serum. This report represents the first demonstration of a pertussis-toxin-substrate-mediated response in this cell line and provides further evidence that G-proteins other than Gi can be functionally inactivated by pertussis toxin.

scholarly journals Sindrom McCune Albright Dengan Manifestasi Fraktur Berulang

2021 ◽  
Vol 36 (1) ◽  
pp. 24-32
Author(s):  
Ruth Nadya ◽  
Frida Soesanti
Keyword(s):  
Vitamin D ◽  
Fibrous Dysplasia ◽  
Guanine Nucleotide ◽  
Polyostotic Fibrous Dysplasia ◽  
Guanine Nucleotide Binding Protein ◽  
Α Subunit ◽  
Mccune Albright Syndrome ◽  
Guanine Nucleotide Binding ◽  
Albright Syndrome ◽  
Mccune Albright

Abstrak Sindrom McCune-Albright (SMA) merupakan kelainan genetik kompleks yang ditandai dengan trias displasia fibrosa poliostotik, café-au-lait, dan hiperfungsi endokrin. Sindrom ini termasuk penyakit langka dengan prevalens sebesar 1 per 100.000 hingga 1.000.000 populasi. Mutasi somatik sporadik gen GNAS (Guanine Nucleotide binding protein Alpha Stimulating) pada kromosom 20q13, yang terjadi pada sindrom ini, mengakibatkan aktivasi G protein α-subunit (Gsα) berkepanjangan yang meningkatkan aktivitas dan fungsi sel terkait. Manifestasi tersering yang ditemukan pada pasien adalah displasia fibrosa (pada 98% kasus). Kasus adalah seorang anak lelaki, 10 tahun, dengan manifestasi fraktur berulang sejak usia 1 tahun dan deformitas tulang. Pemeriksaan bone survey menunjukkan gambaran ground glass dengan lesi litik-sklerotik pada hampir semua tulang yang sesuai dengan displasia fibrosa poliostotik. Pasien ditata laksana dengan pemberian sediaan fosfat, kalsium, serta vitamin D dalam bentuk aktif dan analog. Pemberian bisfosfonat bertujuan untuk mengurangi nyeri tulang dan risiko fraktur pada pasien. Pemantauan berkelanjutan diperlukan untuk mengevaluasi keterlibatan organ endokrin pada pasien dengan SMA.  Kata kunci: displasia fibrosa, fraktur, sindrom McCune Albright Abstract McCune-Albright syndrome (MAS) is a rare complex genetic disorder marked by the triad of polyostotic fibrous dysplasia, café-au-lait and endocrine hyperfunction, affecting 1 in 100.000 to 1.000.000 population. The sporadic somatic mutation of Guanine Nucleotide Binding Protein Alpha Stimulating (GNAS) gene at chromosome 20q13 is the proposed cause of this syndrome, triggering the prolonged activation of  G protein α-subunit (Gsα), which increases the activity and function of cells. The most common clinical manifestation is fibrous dysplasia, occurring in 98% cases. This case occurred in a 10-year-old boy with recurrent fractures since the age of 1-year-old and skeletal deformities. The bone survey examination shows ground glass appearance with multiple sclerotic-lytic lesions on almost every bone, accordingly to the polyostotic fibrous dysplasia. The pasien has been treated with oral phosphate, calcium and vitamin D. Intravenous bisphosphonates was administered to relieve the associated bone pain and reduce the risk of recurring fractures. Longitudinal observation is necessary for a long term monitoring to evaluate the endocrinopathy associated with MAS.  Keywords: fibrous dysplasia, fractures, McCune-Albright syndrome,

scholarly journals Antibodies raised against a peptide corresponding to a Gs alpha domain reveal a vimentin-associated protein.

1993 ◽  
Vol 41 (5) ◽  
pp. 709-717 ◽  
Author(s):  
G Anglade ◽  
P Dupouey ◽  
D Ensergueix ◽  
B Ceard ◽  
A Monneron
Keyword(s):  
Cdna Sequence ◽  
Plasma Membranes ◽  
Ultrastructural Level ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Western Blots ◽  
Weak Staining ◽  
Guanine Nucleotide Binding ◽  
Gs Alpha ◽  
Optical Level

In an attempt to localize the guanine nucleotide binding protein Gs alpha by immunocytochemistry, we synthetized peptides corresponding to several stretches of residues deduced from the published cDNA sequence of Gs alpha and raised antibodies against them. Among the peptides, one corresponding to residues 367-381 elicited antibodies that immunocytochemically detected, at the optical level, what appeared to be vimentin in several cells and tissues. Studies at the ultrastructural level confirmed this observation and also showed weak staining of some areas of plasma membranes of glial and nerve cells. Analysis by Western blots of rat brain subcellular fractions indicated that: (a) the protein stained by the anti-peptide antibodies was associated with the cytoskeleton; and (b) it was not vimentin but a protein of higher molecular weight, 65 KD. We accordingly suggest that the Gs alpha-derived peptide elicited two types of antibodies, one recognizing Gs alpha in fixed tissues, the other recognizing an epitope located in a vimentin-associated protein. This study emphasizes the caution that is needed when conclusions are drawn on the basis of immunocytochemical studies using antipeptide antibodies.

Dual function of Ras in Raf activation

Development ◽  
1998 ◽  
Vol 125 (24) ◽  
pp. 4999-5008 ◽  
Author(s):  
W. Li ◽  
M. Melnick ◽  
N. Perrimon
Keyword(s):  
Plasma Membrane ◽  
Nucleotide Binding ◽  
Dual Function ◽  
Guanine Nucleotide ◽  
Guanine Nucleotide Binding Protein ◽  
Raf Kinase ◽  
P21 Ras ◽  
Guanine Nucleotide Binding ◽  
Drosophila Embryos

The small guanine nucleotide binding protein p21(Ras) plays an important role in the activation of the Raf kinase. However, the precise mechanism by which Raf is activated remains unclear. It has been proposed that the sole function of p21(Ras)in Raf activation is to recruit Raf to the plasma membrane. We have used Drosophila embryos to examine the mechanism of Raf (Draf) activation in the complete absence of p21(Ras) (Ras1). We demonstrate that the role of Ras1 in Draf activation is not limited to the translocation of Draf to the membrane through a Ras1-Draf association. In addition, Ras1 is essential for the activation of an additional factor which in turn activates Draf.

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