Related Experiment Video
Updated: Aug 1, 2026
Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets
Published on: June 25, 2012
Decreased transcript expression coincident with impaired glycosylation in the beta2-adrenergic receptor gene does not
R J Hughes1, M Pasillas, J Saiz
1Department of Pharmacology, University of California at San Diego, La Jolla 92093-0636, USA.
Abstract:
Variants of the S49 mouse lymphoma cell line exhibit multiple lesions along the pathway of cyclic AMP generation in response to beta2-adrenergic stimulation. Two such variants, beta(p) and beta(d), are characterized by decreased receptor binding and mRNA expression, 50% and 25% of wild-type receptor expression, respectively. The rate of beta2-adrenergic receptor synthesis was measured and found to be decreased in the beta d cells vis-a-vis the rate in wild type cells. The molecular mass of the beta2-adrenergic receptor in the S49 wild-type, beta(p) and beta(d) variant cells was estimated by labeling the receptor with the photoaffinity probe [125I]iodocyanopindololdiazirine. Receptor size was found to be 67,000 and 47,000 Da in the wild-type and 60,000 and 42,000 in the two variant cells. This 6 kDa discrepancy in mass was abolished upon treatment of labeled cell extracts with N-glycosidase F, suggesting the possibility of either N-terminal truncation or altered glycosylation of the receptor in the variant cells. To distinguish between these possibilities, we sequenced the beta2-adrenergic receptor gene and two kilobases of the 5'-non-coding region. No differences were found in the coding region of the gene from wild-type, beta(p) and beta(d) S49 cells suggesting that both the diminished expression and the decreased size of beta2-adrenergic receptor in the beta(p) and beta(d) S49 variants are related to impaired glycosylation of the receptor. This hypothesis was substantiated by the reduced retention of the variant cells' beta2-adrenergic receptor on immobilized WGA. Furthermore, growth of the S49 cells in the presence of the alpha-mannosidase II inhibitor, swainsonine, preferentially impaired the ability of the receptors derived from the variant cells to bind to WGA. These results imply that altered expression and glycosylation of G-protein-linked receptors occur as a consequence of one or more mutations outside the receptor's open reading frame.
Insights
Variants of the S49 mouse lymphoma cell line show altered beta2-adrenergic receptor expression and size. Impaired glycosylation, not gene mutations, causes these changes, impacting cyclic AMP generation.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- S49 mouse lymphoma cell line variants (beta(p), beta(d)) exhibit defects in beta2-adrenergic receptor-mediated cyclic AMP generation.
- These variants display reduced receptor binding and mRNA expression compared to wild-type cells.
Purpose of the Study:
- To investigate the molecular basis for the altered beta2-adrenergic receptor expression and size in S49 cell variants.
- To determine if genetic mutations or post-translational modifications, specifically glycosylation, are responsible for the observed receptor defects.
Main Methods:
- Quantitative analysis of receptor binding and mRNA expression.
- Measurement of beta2-adrenergic receptor synthesis rates.
- Molecular mass estimation using photoaffinity labeling and N-glycosidase F treatment.
- Sequencing of the beta2-adrenergic receptor gene and 5'-non-coding region.
- Wheat germ agglutinin (WGA) affinity chromatography and swainsonine treatment.
Main Results:
- Beta(p) and beta(d) variants showed 50% and 25% of wild-type receptor expression, respectively, with decreased synthesis rates in beta(d) cells.
- Receptor molecular mass differed between variants and wild-type, with a discrepancy resolved by N-glycosidase F treatment.
- No mutations were found in the coding or 5'-non-coding regions of the beta2-adrenergic receptor gene.
- Variant receptors exhibited reduced WGA binding, further impaired by swainsonine, indicating altered glycosylation.
Conclusions:
- Diminished expression and size of beta2-adrenergic receptors in S49 variants are attributed to impaired glycosylation, not alterations in the receptor gene sequence.
- Mutations outside the receptor's open reading frame likely affect G-protein-linked receptor expression and glycosylation.
More Related Videos
09:21Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity
Published on: October 22, 2018
09:41Measuring the Rate of Lipolysis in Ex Vivo Murine Adipose Tissue and Primary Preadipocytes Differentiated In Vitro
Published on: March 17, 2023
Related Concept Videos
01:42What is Gene Expression?
Gene expression is the process in which DNA directs the synthesis of functional products, that is, proteins. Cells can regulate gene expression at various stages. It allows organisms to generate different cell types and enables cells to adapt to internal and external factors.
Genetic Information Flows from DNA to RNA to Protein
A gene is a stretch of DNA that serves as the blueprint for functional RNAs and proteins. Since DNA is made up of nucleotides and proteins consist of amino...
01:58Cell Specific Gene Expression
01:36What is Gene Expression?
01:30General Transcription Factors
01:26Adrenergic Receptors: β Subtype
Isoprenaline > Adrenaline > Noradrenaline
Neurotransmitter binding to these receptors causes activation of adenylyl cyclase resulting in increased concentrations of cAMP and modulation of calcium ion channels within the cell. They are further classified into β1, β2, and β3 subtypes.
β1-adrenoceptors: β1-adrenoceptors have equal affinities for...
01:11Pharmacogenetics of Drug Targets: β₂-Adrenergic Receptors, Apo E, Thymidylate Synthase