Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells.

Juhnn, Yong Sung; Jones, Teresa L.Z.; Spiegel, Allen M.

doi:10.1083/jcb.119.3.523

Cited by 21 publications

(15 citation statements)

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“…These results are consistent with our previous findings that no single region of Gsα, including the amino terminal region, was solely responsible for membrane binding in COS-1 cells (Juhnn et al, 1992(Juhnn et al, , 1993. However, our results do not support the model of activation coupled depalmitoylation with the release of G sα to cytosol (Wedegaertner et al, 1993; We d e g a e r t n e r and Bourne, 1994).…”

Section: Discussionsupporting

confidence: 83%

“…The endogenous forms of Gsα exhibited two groups of bands on western blots when the blot was visualized with an antibody against Gsα, RM (Juhnn et al, 1992). The larger slow moving immunoreactivity increased in the cells transfected with wild type or mutant forms of Gsα cDNA demonstrating respective overexpression in the COS-1 cells (Figure 1a).…”

Section: Palmitoylation Of the Mutant Gs Expressed In Cos-1 Cellsmentioning

confidence: 97%

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Retainment of membrane binding capacity of non-palmitoylated Gs α mutants expressed in COS-1 cells

Yang

Cho²,

Bae³

et al. 1998

Exp Mol Med

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IntroductionHeterotrimeric guanine nucleotide binding regulatory proteins (G proteins) transduce extracellular signals such as hormones, neurotransmitters and photons into intracellular signals by receptor-effector coupling. When the G protein binds to the ligand-activated receptor, it replaces the bound GDP with GTP. The GTP-bound activated G protein regulates effectors such as enzymes and ion channels (Gilman, 1987;Kaziro et al., 1991; Neer, 1995). Because most of the receptors coupled to G proteins are integral proteins that have seven hydro-phobic transmembrane domains, the G proteins need to have a membrane binding capacity to receive signals from the membrane receptors. The α subunit of G protein is known to bind tightly to the cytoplasmic face of the plasma membrane without any membrane spanning hydrophobic domain. The β γ subunit complex has a high affinity for the membrane because of isoprenylation of the gamma subunit, and plays an important role in anchoring the α subunit of G proteins (Sternweis, 1986). However, fatty acylation with myristic acid and palmitoylation was found to be important for membrane binding by α subunits of the G protein family (Jones et al., 1990, Mumby, 1997. The reversible and dynamic palmitoylation had been claimed to be critical in membrane binding by the alpha s u b u n i t of the stimulatory G protein (Gsα) (Wedegaertner et al., 1993;Wedegaertner and Bourne, 1994), but it was also argued that this domain of Gsα was not essential for membrane binding from the finding that Gsα m u t a n t s deleted in this domain retained membrane binding capacity (Degtyarev et al. , 1993;Juhnn, 1993). Thus it is necessary to make clear the importance of this modification in the binding of Gsα.The present study was performed to evaluate the role of palmitoylation in membrane binding of Gsα. We constructed Gsα mutants by replacing the glycine-2, the cognate site of myristoylation in Giα with alanine, and cysteine-3, the site of palmitoylation with serine, and then analyzed the intracellular distribution of the mutant proteins expressed in COS-1 cells by quantitative EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 30, No 4, 235-239, December 1998 Jung Abbreviations: G protein, guanine nucleotide-binding regulatory protein; Gi, adenylyl cyclase inhibitory G protein; Gs, adenylyl cyclase stimulatory G protein; Gsα, α subunit of Gs; Gα,α subunit of G protein ; Gβγ, βγ subunit of G protein Retainment of membrane binding capacity of nonpalmitoylated Gs mutants expressed in COS-1 cells

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Section: Discussionsupporting

confidence: 83%

Section: Palmitoylation Of the Mutant Gs Expressed In Cos-1 Cellsmentioning

confidence: 97%

Retainment of membrane binding capacity of non-palmitoylated Gs α mutants expressed in COS-1 cells

Yang

Cho²,

Bae³

et al. 1998

Exp Mol Med

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“…Immunoblot analysis was performed as described previously using antibodies speci¢c to GKs (RM) [14], GKi (AS7), GKq (QL), GL (SW) [15,16], HA (Roche Applied Science), or actin (Santa Cruz). Band densities were quanti¢ed with an image analyzer (Model GS-700, Bio-Rad), and expressed as a ratio to the corresponding band density in non-transfected CHO cells or vector-transfected cells.…”

Section: Immunoblot Analysismentioning

confidence: 99%

Coordinate expression of the α and β subunits of heterotrimeric G proteins involves regulation of protein degradation in CHO cells

et al. 2003

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Individual cell types express a characteristic balance between heterotrimeric G protein K K and LQ LQ subunits, but little is known about the regulatory mechanism. We systemically examined the regulatory mechanism in CHO cells. We found that expression of GK Ks, GK Ki2, and GK Kq proteins increased in direct proportion to the increase of GL L1Q Q2 overexpressed transiently. Expression of GL L protein also increased following overexpression of GK Ks, GK Ki2, and GK Kq. The GLQ LQ overexpression stimulated degradation of GL L in contrast to reduction of GK Ks degradation. We conclude that coordinate expression of the G protein subunits involves regulation of protein degradation via proteasome in CHO cells. ß

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“…Some a subunits (a,) are not myristoylated and these must be held in the membrane in other ways. The a, subunit, for example, may be anchored through its carboxyl-terminus (Audigier et al, 1990), although there is conflicting evidence on this point (Juhnn et at., 1992). Myristoylation of a subunits affect two key functions: association with the membrane and with @y. Alteration of either is likely to have major consequences for signal transduction.…”

Section: Interaction Of a Subunits With Membranesmentioning

confidence: 99%

G proteins: Critical control points for transmembrane signals

Neer

1994

Protein Science

121

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Heterotrimeric GTP-binding proteins (G proteins) that are made up of alpha and beta gamma subunits couple many kinds of cell-surface receptors to intracellular effector enzymes or ion channels. Every cell contains several types of receptors, G proteins, and effectors. The specificity with which G protein subunits interact with receptors and effectors defines the range of responses a cell is able to make to an external signal. Thus, the G proteins act as a critical control point that determines whether a signal spreads through several pathways or is focused to a single pathway. In this review, I will summarize some features of the structure and function of mammalian G protein subunits, discuss the role of both alpha and beta gamma subunits in regulation of effectors, the role of the beta gamma subunit in macromolecular assembly, and the mechanisms that might make some responses extremely specific and others rather diffuse.

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Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells.

Abstract: Abstract. To elucidate the structural basis for mem-

Cited by 21 publications

References 39 publications

Retainment of membrane binding capacity of non-palmitoylated Gs α mutants expressed in COS-1 cells

Retainment of membrane binding capacity of non-palmitoylated Gs α mutants expressed in COS-1 cells

Coordinate expression of the α and β subunits of heterotrimeric G proteins involves regulation of protein degradation in CHO cells

G proteins: Critical control points for transmembrane signals

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