Dimerization properties of the transmembrane domains of Arabidopsis CRINKLY4 receptor-like kinase and homologs

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Abstract

CRINKLY4 (CR4) is a plant serine–threonine receptor kinase. In Zea mays, CR4 functions in the differentiation of the leaf epidermis and the aleurone cell layer and, in Arabidopsis thaliana, the ortholog ACR4 is involved in the development of the integument and seed coat. The Arabidopsis genome also encodes four CR4-related proteins (CRR) whose functions are not known. Based on studies of animal receptor kinase proteins it is likely that the molecular basis of function of CR4 and related proteins is mediated by receptor dimerization. The importance of the transmembrane (TM) domain in the dimerization of several receptor kinases has been demonstrated by the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment. In this study, we have used the TOXCAT assay to investigate the potential of the CR4 and CR4-related TM domains to homo-dimerize. Our investigation indicates that the CR4 TM domain and the CRR TM domains have higher propensities for homo-dimerization than the ACR4 TM domain. Interestingly, the dimerization potential of the ACR4 TM domain is significantly weaker even though 13 of 24 amino acids are identical to that of the CR4 TM domain. In order to determine the contributions of specific amino acids to the higher dimerization potential of CR4 compared to ACR4, mutations were made at specific sites in ACR4 TM domain and the strength of the dimer assessed by the TOXCAT assay. One mutation restored the activity to the CR4 level, while other mutations produced either no change or significantly increased the dimerization potential of the ACR4 TM domain. Our results indicate that the TM domains of CR4, ACR4 and the CRR receptor family of proteins have the intrinsic capacity to homo-dimerize, albeit with varying degrees of affinity.

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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