Internal Sequences from Proteins Digested in Polyacrylamide Gels

The regulator of G protein signaling (RGS)-9-1⅐G␤5 complex forms the GTPase accelerating protein for G␣t in vertebrate photoreceptors. Although the complex is soluble when expressed in vitro, extraction of the endogenous protein from membranes requires detergents. The detergent extracts contain a complex of RGS9-1, G␤5, G␣t, and a 25-kDa phosphoprotein, R9AP (RGS9-1-Anchor Protein). R9AP is encoded by one intronless gene in both human and mouse. Full or partial cDNA or genomic clones were obtained from mice, cattle, human, zebrafish, and Xenopus laevis. R9AP mRNA was detected only in the retina, and the protein only in photoreceptors. R9AP binds to the N-terminal domain of RGS9-1, and anchors it to the disk membrane via a C-terminal transmembrane helix.T o ensure high efficiency of phototransduction, the essential components of the phototransduction cascade are packed at a high density on photoreceptor outer segment disk membranes (1), where phototransduction occurs. One challenge in photoreceptor biology has been to determine the mechanisms by which these molecules and their modulators are localized to the disk membranes. This question has been particularly puzzling for regulator of G protein signaling (RGS)-9-1, which is the GTPase accelerating protein (GAP) for the visual G protein G ␣t (2-4).RGS9-1 plays an essential role in setting the timing of the recovery phase of light responses in vertebrate photoreceptors (5, 6). It is a tightly bound peripheral membrane protein (7), but has no obvious features that target it to membranes and is largely soluble when expressed in vitro (8, 9). RGS9-1 forms a constitutive complex with its obligate subunit, G ␤5 (5, 8, 10), and it also interacts with the inhibitory subunit of the effector for G ␣t , cGMP phosphodiesterase-␥ (PDE␥) (11)(12)(13)(14). Neither G ␤5 nor PDE␥ can serve as its membrane anchor, because selective removal of either of them does not affect RGS9-1 membrane binding (2, 9, 15). Closely related proteins like RGS7 and the RNA processing variant RGS9-2, are found in the cytoplasm or nucleus (16-18), as well as on plasma membranes. Other RGS proteins in general show highly varied distribution patterns (19)(20)(21)(22)(23)(24)(25)(26). Variable subcellular targeting of RGS proteins may be a mechanism for regulation of their functions (18,(27)(28)(29).We describe here the identification by coimmunoprecipitation of a heterotetrameric complex containing RGS9-1, G ␤5L , G ␣t , and a previously unknown photoreceptor-specific protein, R9AP. R9AP interacts with the N-terminal domain of RGS9-1 and has a transmembrane helix at its C terminus, allowing it to serve as the RGS9-1-anchor protein. Materials and MethodsBuffers. The following standard buffers were used: low-salt buffer (5 mM Tris⅐HCl͞0.5 mM MgCl 2 ), GAPN buffer (10 mM Hepes͞ 100 mM NaCl͞2 mM MgCl 2 ), high-salt buffer (10 mM Hepes͞1 M NH 4 Cl͞2 mM MgCl 2 ), urea buffer (4 M urea͞5 mM Tris⅐HCl). The pH of all buffers was adjusted to 7.4-7.5, and 1 mM DTT and Ϸ20 mg/liter solid PMSF were added before use.P...

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“…In-gel proteolytic digestion was carried out as described (35). The proteolytic peptides were separated by 18% tricine SDS͞PAGE (36).…”

Section: Methodsmentioning

confidence: 99%

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

Wensel²

2002

Proc. Natl. Acad. Sci. U.S.A.

157

150

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“…Lys-C is a serine protease and, at pH 8.3, specifically cleaves peptide bonds C-terminally at lysine [16]. After the action of this enzyme, unglycated and glycated HSA give the MALDI mass spectra of Figure 4a and b respectively.…”

Section: Digestion By Lys-cmentioning

confidence: 99%

Enzymatic digestion and mass spectrometry in the study of advanced glycation end products/peptides

Lapolla

Fedele

Reitano

et al. 2004

J. Am. Soc. Mass Spectrom.

159

151

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An extensive study was carried out on HSA and non-enzymatically glycated HSA by enzymatic digestion with trypsin and endoproteinase Lys-C, with the aim of identifying specific glycated peptides deriving from enzymatic digestion of glycated HSA. They may be considered, in pectore, as advanced glycation end products/peptides. These compounds, important at a systemic level in diabetic and nephropathic subjects, are produced by enzymatic digestion of in vivo glycated proteins: They are related to the pathological state of patients and have been invoked as responsible for tissue modifications. The digested mixtures obtained by the two enzymes were analyzed by MALDI/MS and LC/ESI/MS n , and clear cut differences were found. First of all, the digestion products of glycated HSA are generally less abundant than those observed in the case of unglycated HSA, accounting for the lower proclivity of the former to enzymatic digestion. MS/MS experiments on doubly charged ions, comparisons with a protein database, and molecular modeling to identify the lysine NH 2 groups most exposed to glycation, identified some glycated peptides in digestion mixtures obtained from both types of enzymatic digestion. Residues 233 K, 276 K, 378 K, 545 K, and 525 K seem to be privileged glycation sites, in agreement with the fractional solvent accessible surface values calculated by molecular modeling. (J Am Soc Mass Spectrom 2004, 15, 496 -509)

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“…In-gel digestion with trypsin was performed according to published methods (Jeno et al, 1995;Shevchenko et al, 1996;Wilm et al, 1996) modified for use with a robotic digestion system (Investigator ProGest, Genomic Solutions) (Wait et al, 2001). Silver-stained gel pieces were first washed with 30 ml of 15 mM potassium ferricyanide/50 mM sodium thiosulphate (Gharahdaghi et al, 1999), followed by successive rinses with deionized water, 50 mM ammonium hydrogen carbonate buffer and acetonitrile.…”

Section: Tandem Mass Spectrometrymentioning

confidence: 99%

Androgens target prohibitin to regulate proliferation of prostate cancer cells

et al. 2004

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Proteins involved in the growth response of prostate cancer cells to androgen were investigated by comparing the proteomes of LNCaP cells treated with vehicle or androgen. Whole-cell lysates were separated by twodimensional PAGE, and HPLC-MS/MS was used to identify androgen-regulated proteins. Prohibitin, a protein with cell-cycle regulatory activity, was shown to be downregulated by 50% following androgen stimulation. Western blot and reverse transcription-PCR experiments confirmed the result and showed that regulation occurs at the level of transcription. To determine the importance of prohibitin in androgen-stimulated growth, we used transient transfection to overexpress the protein and RNA interference to knock down the protein. Subsequent FACS analysis showed that cells with reduced levels of prohibitin showed a slight but reproducible increase in the percentage of population in cell cycle, while cells with increased prohibitin levels showed a clear reduction in the percentage entering cell cycle, following dihydrotestosterone stimulation, when compared to untransfected controls. Confocal microscopy showed localization of prohibitin in the nucleus as well as the mitochondria of LNCaP cells. It therefore seems that the regulation of prohibitin is a vital part of the cellular growth response to androgen stimulation in LNCaPs and prohibitin may have a nuclear regulatory role in cell-cycle progression.

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Internal Sequences from Proteins Digested in Polyacrylamide Gels

Cited by 202 publications

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R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

R9AP, a membrane anchor for the photoreceptor GTPase accelerating protein, RGS9-1

Enzymatic digestion and mass spectrometry in the study of advanced glycation end products/peptides

Androgens target prohibitin to regulate proliferation of prostate cancer cells

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