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Establishing and functional characterization of an HEK-293 cell line expressing autofluorescently tagged β-actin (pEYFP-ACTIN) and the neurokinin type 1 receptor (NK1-R)

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Abstract

This study focused on establishing and making a comprehensive functional characterization of an HEK-293-transfected cell line that would coexpress the enhanced yellow fluorescent protein-actin (pEYFP-actin) construct and the neurokinin type 1 receptor (NK1-R), which is a member of the seven transmembrane (7TM) receptor family. In the initial selection procedure, the cloning ring technique was used alone, but failed to yield clones with homogenous pEYFP-actin expression. Flow cytometry sorting (FCS) was subsequently used to enrich the pEYFP-actin-expressing subpopulation of cells. The enzyme-linked immunosorbent assay (ELISA), FCS and quantitative real-time reverse transcription/polymerase chain reaction (RT-PCR) were then employed to monitor the passage-dependent effects on transgene expression and to estimate the total β-actin/pEYFP-actin ratio. NK1-R was characterized via radioactive ligand binding and the second messenger assay. The suitability of the pEYFP-actin as a marker of endogenous actin was assessed by colocalizing pEYFP-actin with rhodamine-phalloidine-stained F-actin and by comparing receptor- and jasplakinolide-induced changes in the actin cytoskeleton organization. These experiments demonstrated that: i) both constructs expressed in the generated transfected cell line are functional; ii) the estimated pEYFP-actin: endogenous β-actin ratio is within the limits required for the functional integrity of the actin filaments; and iii) pEYFP-actin and rhodamine-phalloidine-stained F-actin structures colocalize and display comparable reorganization patterns in pharmacologically challenged cells.

Abbreviations

7TM:

seven transmembrane receptor

Bmax :

max. receptor number

BSA:

bovine serum albumin

Ct:

threshold cycle

DMEM:

Dulbecco’s modified Eagles’s Medium

DNA:

deoxyribonucleic acid

D-PBS:

Dulbecco’s phosphate-buffered saline

ELISA:

enzyme-linked immunosorbent assay

F-actin:

filamentous actin

FCA:

flow cytometry analysis

FCS:

flow cytometry sorting

GFP:

green fluorescent protein

GPCR:

G-protein coupled receptor

HA:

hemagglutinin

HANK1-R:

N-terminally HA-tagged human neurokinin-1 receptor

HEK-293:

human embryonic kidney cells

HIFCS:

heat inactivated fetal calf serum

HRP:

horseradish peroxidase

IC50 :

50% inhibitory concentration

IP1:

inositol phosphate 1

mRNA:

messenger ribonucleic acid

NK1-R:

neurokinin type 1 receptor

pEYFP:

enhanced yellow fluorescent protein

PN:

passage number

RT:

reverse transcription

RT-PCR:

quantitative real-time reverse transcription / polymerase chain reaction

SP:

substance P

TMB:

tetramethylbenzidine

WT:

wildtype

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Correspondence to Milka Vrecl.

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Key words

  • Cytoskeleton
  • Actin filaments
  • HEK-293
  • Neurokinin type 1 receptor
  • Flow cytometry
  • Confocal microscopy