Published: June 15th, 2013
Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method—adapted from a coculture model of synaptogenesis—for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.
Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.
We describe here a method to facilitate investigations of how the extracellular domains of specific transmembrane proteins affect insulin secretion. The method probes the effects of interactions of the protein of interest with proteins (or possibly other molecules) on the pancreatic beta-cell surface. The method allows investigations of how cell-surface proteins expressed by beta cells or by other neighboring cells (e.g. endothelial cells, neurons, pancreatic alpha cells) affect beta-cell function through transcellular interactions (i.e. through interactions with interaction partners on the surface of adjacent beta cells).
1. Transfection of HEK293 Layer
Using the method described here, we have tested the effect of different variants of the protein neuroligin on insulin secretion. This complements our published work investigating the effect of neuroligin-2 on beta-cell function10. Figure 2, for example, depicts results obtained from coculturing INS-1 beta cells with HEK293 cells transfected to express a neuroligin isoform referred to here as NL-X. This experiment was designed to test the hypothesis that NL-X engages in transcellular interactio.......
The coculture method described here provides an effective way to determine the physiological importance of specific beta-cell-surface, transmembrane proteins, and specifically of their extracellular domains. By culturing beta cells or insulinoma cells (such as the INS-1 cells employed here) in contact with HEK293 cells displaying a protein of interest on the cell surface, experiments can be designed to determine the effects of extracellular protein-protein interactions without directly disturbing the intracellular milieu.......
This work was supported by National Institutes of Health grant R01DK080971 and Juvenile Diabetes Research Foundation grant 37-2009-44. We also appreciate support received from the UCSD Pediatric Diabetes Research Center (PDRC).....
|Name of the reagent
|pcDNA 3.3 vector/backbone
|Electron Microscopy Sciences
|Fetal bovine serum
|RIPA lysis buffer
Copyright © 2024 MyJoVE Corporation. All rights reserved