The Gastrin-releasing Peptide Receptor Is Rapidly Phosphorylated by a Kinase Other Than Protein Kinase C After Exposure to Agonist

Kroog, Glenn; Sainz, Eduardo; Worland, Peter J.; Akeson, Mark; Benya, Richard V.; Jensen, Robert T.; Battey, James F.

doi:10.1074/jbc.270.14.8217

Cited by 46 publications

(49 citation statements)

References 48 publications

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“…However, such ®ndings are not universal. A series of receptors have been observed to be phosphorylated by phorbol esters and their respective agonists, but inhibition of PKC activity was found only to attenuate the e ect of the phorbol esters (Ali et al, 1993;Tobin & Nahorski, 1993;Ishii et al, 1994;Lattion et al, 1994;Kroog et al, 1995). These studies therefore concluded that the mechanism underlying agonist-induced phosphorylation did not include PKC.…”

Section: Receptor Phosphorylation By Angiotensin IImentioning

confidence: 89%

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT_1A receptor

Balmforth

Shepherd

Warburton

et al. 1997

British J Pharmacology

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1 The role of protein kinase C (PKC) in the mechanism underlying rapid agonist-induced desensitization of angiotensin AT 1 receptors remains unresolved. A major problem has been to isolate these receptors in a su ciently puri®ed form to allow study of their phosphorylation state. 2 A cleavable (His) 6 a nity tag was introduced into the N-terminus of the recombinant AT 1A receptor and stably expressed in human embryonic kidney cells. This a nity tag allowed rapid isolation, puri®cation and determination of the phosphorylation state of the AT 1A receptor. Using these cells, we determined the role of PKC in both agonist-induced receptor phosphorylation and desensitization under identical conditions. 3 Agonist-induced phosphorylation of the AT 1A receptor was observed at both low and high concentrations of angiotensin II (AII). Preincubation of cells with Ro-31-8220 (a PKC speci®c inhibitor) revealed that at low concentrations of AII (1 nM), PKC appeared to be the main kinase involved in receptor phosphorylation. In contrast, at high concentrations of AII (100 nM), although PKC-mediated phosphorylation of the receptor was observed, this was overshadowed by a second kinase. 4 In preliminary desensitization studies we observed that at a low concentration of AII, preincubation with Ro-31-8220 attenuated desensitization, whilst at high concentrations of AII (100 nM) it had little or no e ect on the level of desensitization observed.5 These data directly demonstrate an association between PKC-induced receptor phosphorylation and desensitization at low concentrations of AII. Since circulating concentrations of AII are in the picomolar range, we propose that PKC is the physiologically relevant mediator of AT 1 receptor desensitization.

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Section: Receptor Phosphorylation By Angiotensin IImentioning

confidence: 89%

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT_1A receptor

Balmforth

Shepherd

Warburton

et al. 1997

British J Pharmacology

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“…Antibody against the GRPR (Kroog et al 1995) was obtained from Dr. James Battey (NIDCD, NIH; Bethesda, MD). All immunohistochemical reagents, including Large Volume DAKO LSAB(R)2 Kit and DAKO Liquid DAB SubstrateChromogen System were from DAKO (Carpinteria, CA).…”

Section: Reagentsmentioning

confidence: 99%

Quantitative Immunohistochemistry by Measuring Cumulative Signal Strength Using Commercially Available Software Photoshop and Matlab

Matkowskyj

Schonfeld

Benya

2000

J Histochem Cytochem.

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134

120

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SUMMARYCurrently available techniques for performing quantitative immunohistochemistry (Q-IHC) rely upon pixel-counting algorithms and therefore cannot provide information as to the absolute amount of chromogen present. We describe a novel algorithm for true Q-IHC based on calculating the cumulative signal strength, or energy, of the digital file representing any portion of an image. This algorithm involves subtracting the energy of the digital file encoding the control image (i.e., not exposed to antibody) from that of the experimental image (i.e., antibody-treated). In this manner, the absolute amount of antibody-specific chromogen per pixel can be determined for any cellular region or structure. Q uantitative immunohistochemistry (Q-IHC) in the predigital era depended on the observations of multiple investigators using an arbitrary scale to grade the extent and intensity of chromogen present (Shi et al. 1991(Shi et al. ,1993. This approach was inherently limited by observer subjectivity and bias, by inter-and intraobserver variation, and generated data of limited range (i.e., amount is usually quantified on a 0-4 scale). With the advent of digital photomicroscopy, however, these weaknesses could in theory be eliminated and true quantification achieved.Early attempts at Q-IHC involved converting analog images into a digital format and then transforming the 256 separate shades of red, green and blue that are obtained when working in 24-bit RGB color to singlechannel grayscale (Mosedale et al. 1996). The area of interest is defined and the mean gray level of the selected pixels determined. Later attempts at Q-IHC employed color thresholding using commercial software (i.e., Adobe Photoshop) (Fermin and Degraw 1995), followed by counting the total number of pixels of appropriate value (Lehr et al. 1997(Lehr et al. ,1999Ruifrok 1997). For example, in DAB-based immunohistochemistry all the pixels containing "brown" within a prespecified spectral range are identified in Photoshop using the Magic Wand tool. The total numbers of pixels identified are then counted using the histogram function (Lehr et al. 1999). Although these algorithms represent significant improvements over traditional methods for evaluating analog images, these approaches still do not allow true Q-IHC to be performed.Determining the absolute amount of chromogen present necessitates calculating the cumulative signal strength of the image being evaluated. This can be done only by calculating signal energy, E, which is defined in the mathematical (Jain 1989) and not the physical sense. Here we provide an algorithm for true Q-IHC that relies on calculating the energy of images captured in Photoshop using a high-resolution digital camera and then processing the image's unmodified and full digital file using the powerful enabler language Matlab. To demonstrate this algorithm, we studied the gastrin-releasing peptide receptor (GRPR) aberrantly expressed by human colon cancers. Because we have previously shown that human colon cancers variably express this ...

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“…BALB/c 3T3 fibroblasts stably expressing myc-tagged GRPr 29 were transfected with different LPXN constructs using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. Double selection with Hygromycin B (200 mg/ml) and G-418 (300 mg/ml) was begun 48 h after transfection and continued for 2-3 weeks.…”

mentioning

confidence: 99%

Leupaxin is similar to paxillin in focal adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading

Chen

Kroog

2010

Cell Adhesion & Migration

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The Gastrin-releasing Peptide Receptor Is Rapidly Phosphorylated by a Kinase Other Than Protein Kinase C After Exposure to Agonist

Cited by 46 publications

References 48 publications

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT_1A receptor

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT_1A receptor

Quantitative Immunohistochemistry by Measuring Cumulative Signal Strength Using Commercially Available Software Photoshop and Matlab

Leupaxin is similar to paxillin in focal adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading

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The Gastrin-releasing Peptide Receptor Is Rapidly Phosphorylated by a Kinase Other Than Protein Kinase C After Exposure to Agonist

Cited by 46 publications

References 48 publications

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT1A receptor

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT1A receptor

Quantitative Immunohistochemistry by Measuring Cumulative Signal Strength Using Commercially Available Software Photoshop and Matlab

Leupaxin is similar to paxillin in focal adhesion targeting and tyrosine phosphorylation but has distinct roles in cell adhesion and spreading

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Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT_1A receptor

Evidence of an important and direct role for protein kinase C in agonist‐induced phosphorylation leading to desensitization of the angiotensin AT_1A receptor