Dimerization properties of the transmembrane domains of Arabidopsis CRINKLY4 receptor-like kinase and homologs
Section snippets
General
Media were prepared as described in Sambrook et al. [21]. All cloned TM domains were confirmed by sequence analysis. Unless otherwise stated, ampicillin (AMP) was used at 50 μg/mL and streptomycin (STREP) at 30 μg/mL.
Bacterial strains, vectors, and constructs
TOP10 cells were obtained from Invitrogen. The maltose-binding protein (MBP) deficient Escherichia coli bacterial stain NT326, the expression vector pccKAN, its derivatives pccGpA-WT (encoding the TM domain of wild-type glycophorin A) and pccGpA-G83I (encoding the TM domain of
TOXCAT assay
To study TM dimerization in a natural membrane environment Russ and Engelman [20] developed the TOXCAT system. In this assay, a construct consisting of the N-terminal DNA-binding domain of ToxR (a dimerization dependent transcription factor) fused to the appropriate TM domain and MBP (a monomeric periplasmic protein) is expressed in the inner membrane of NT326 E. coli cells. Association of the TM helices within the membrane induces dimerization of ToxR and transcriptional activation of the
Discussion
The GxxxG motif, containing two glycine residues separated by any three amino acids in a helical template, mediates the dimerization of the TM domain of GpA [30]. It is present in a number of receptors such as members of the epidermal growth factor receptor family [26], the integrins [31] and receptor-like tyrosine phosphatases [32]. It is the most over-represented motif found in a database of TM domain sequences [29] and the most prevalent motif identified in a randomized library created from
Conclusion
Our studies indicate that the dimerization propensities of the maize and Arabidopsis CRINKLY4 TM domains differ significantly. On the other hand, the TM domains of the Arabidopsis CRR proteins are comparable to that of CR4. The difference in ACR4 and CR4 dimerization can be accounted for by a single G438V mutation in ACR4. Other ACR4 mutations vary from causing no change in dimerization potential (A452C) to promoting very strong dimerization (A433F), showing that dimerization potential is
Acknowledgments
We thank Dr. Donald Engelman (Yale University) for providing the NT326 cells and TOXCAT vectors pccKAN, GpA wt and GpA G83I mutant. We also thank Erin Matthews (Yale University) for helpful advice and Dr. Ed Yu of this department for critical reading of the manuscript.
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