Team:Illinois/Antibody GPCR Fusion

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Revision as of 20:30, 13 June 2008

Core Team Members

Dave Luedtke, Bobak Hadidi, Kiruthika Selvadurai, Namita Bakshi, Toni Espina, Sarah Grajdura

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Project Abstract

G protein-coupled receptors, or GPCRs, are transmembrane receptors that sense extracellular objects on the scale of small molecules to large proteins. Activation of the GPCR by ligand binding begins a signal transduction pathway that ultimately results in the transcriptional activation or repression of one or more genes. The signal is transduced with G-proteins, which are signalling proteins that associate with GTP and GDP, as well as kinase cascades. Yeast cells are known to utilize two GPCRs signal transduction pathways, one to detect the presence of glucose and the other to initiate mating. We hope to engineer the well-characterized mating pathway to produce a colorimetric change in the cell upon detecting a novel molecule-- a surface protien of some water-borne pathogen, or possibly a toxin secreted by such a water-borne pathogen. One issue that may have to be dealt with is the cell wall of the yeast: it may prevent the target protein from drawing near enough to the receptor to activate it.

Specific Plans, Supplies, and Protocols

In theory, this is how we will progress:

• Create a fusion protein that links an antibody against cholera toxin to the Ste2 GPCR of S. cereviviae, the pheremone response GPCR.
  • 1)Find the sequences of the GPCR and the antibody
  • 2)Select site of fusion
  • 3)Have the gene sequenced--This will be a lengthy gene. We might be better off somehow obtaining a gene containing the natural GPCR (perhaps from a research group) and inserting the gene of the antibody.
• Express the antibody/GPCR fusion protein in yeast that lack the wild type receptor.
  • The gene will most likely be on a plasmid. We may have to integrate the gene into the yeast's chromosome, however.
• Measure the activation of the GPCR by the toxin (and by the natural pheremone) using a reporter gene.
  • Several studies have used the FUS1 promoter in conjunction with HIS3 selection. The FUS1 promoter seems to be a good choice for our purposes.
• Use site specific directed evolution to increase the effectiveness of the new GPCR.

Alternatively, we might begin looking into replacing the yeast receptor with a mammalian or novel receptor with an affinity for our target protein. If we go down this route, we will also have to replace the G-alpha subunit with a chimeric one that has affinity for both the new receptor and the yeast G-betagamma complex.

To Research

• Cell Wall Issue
  • "The potency of some ligands might be reduced in yeast compared with mammals [for receptors taken from mammalian cells]. Using Gpa1–Ga chimeras probably explains part of this discrepancy [original yeast G-alpha subunits have low affinity for the new receptors and the mammalian G-alpha has low affinity for yeast G-betagamma, we will bypass this problem altogether by fusing the antibody to the original yeast receptor], but another factor might be the ability of ligands to penetrate the yeast cell wall. Potencies for small ligands are similar in yeast and mammals, those for intermediate ligands are about two orders of magnitude less in yeast, and large ligands such as chemokines (polypeptides with 70–80 residues) are often unable to activate receptors when applied exogenously to yeast. The fact that smaller versions of these ligands can be more efficient agonists suggests that accessibility is an issue."[1]
• Antibody Sequence
• Sources of yeast strains deficient in specific genes
• Cholera Toxin or possible target protein

G Proteins and their Receptors

G protein Heterotrimer: Alpha chain is yellow, Beta-Gamma complex is Blue, GDP is black, membrane is shown in grey.

G proteins are comprised of 3 subunits, collectively known as a heterotrimeric or large G protein. The individual pieces are Gα, Gβ and Gγ. When the appropriate molecule is bound to the receptor, the GDP bound to the Gα unit becomes GTP. More conformational changes are induced causing Gβγ and Gα to dissociate. Depending on the signal cascade, either complex, Gα-GTP or Gβγ may now induce the appropriate linked signal pathway.

As opposed to other signal receptors, the ligands that bind GPCRs typically bind within the transmembrane domain. When the receptor is bound by its ligand, it shifts shape, activating the G protein (by allowing the exchang of GDP for GTP). Also, G protein complexes may or may not be coupled with the receptors, instead, if they happen across a receptor with its activating ligand bound, the G protein will be activated (GDP -> GTP) and induce its signal cascade. (wikipedia).

Literature Research and References

[1]Functional analysis of heterologous GPCR signaling pathways in yeast

[2]http://www.nature.com/embor/journal/v2/n7/full/embor385.html

Ste2 information:http://www.bio.davidson.edu/Courses/genomics/2002/Statler/STE2.htm

2 GPCR pathways in yeast: http://www.nature.com/embor/journal/v2/n7/images/embor385-f1.gif

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