Dimerization properties of the transmembrane domains of Arabidopsis CRINKLY4 receptor-like kinase and homologs.
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.
Stokes
Kevin D
KD
Department of Biochemistry, Biophysics and Molecular Biology, 1210 Molecular Biology Building, Iowa State University, Ames, Iowa 50011, USA.
Gururaj Rao
A
A
D017360:Arabidopsis / Q000201:enzymology*; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D007527:Isoenzymes / Q000737:chemistry; D021962:Membrane Microdomains / Q000737:chemistry*; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry*
D007527:Isoenzymes; D010940:Plant Proteins; D011494:Protein Kinases