Biology relies upon the enlarged repertoire of interactions that occur when proteins are posttranslationally modified. In recent years, it has become clear that methyl groups stand beside phosphate groups as major controlling elements in protein function. A wide variety of methylation (and in some cases demethylation) reactions occur at the side chains of a number of amino acid residues and at protein N- and C-termini. These modifications generate distinct sets of chemical interactions that play roles in a multitude of regulatory pathways (Clarke and Tamanoi, 2006
The modification of arginine side chain guanidino groups is probably the most quantitatively extensive protein methylation reaction in mammalian cells (Gary and Clarke, 1998
). The number of distinct modified proteins is also large (Pahlich et al., 2006
). Arginine is unique among amino acids as its guanidino group contains five potential hydrogen-bond donors that are positioned for favorable interactions with biological hydrogen-bond acceptors. In protein-DNA complexes, arginine residues are the most frequent hydrogen bond donors to backbone phosphate groups and to thymine, adenine, and guanine bases (Luscombe et al., 2001
). Specific networks of hydrogen bonds can form with arginine residues and adjacent phosphate groups in RNA loops (Calnan et al., 1991
), and the arginine-aspartate two H-bond interaction is especially stable in proteins (Mitchell et al., 1992
). Each addition of a methyl group to an arginine residue not only changes its shape, but also removes a potential hydrogen bond donor. Such chemistry could promote the preferential inhibition by methylation of some, but not all, binding partners. For example, arginine methylation of the Sam68 proline-rich motifs can inhibit its binding to SH3, but not WW domains (Bedford et al., 2000
). Methylation of arginine residues might also increase their affinity to aromatic rings in cation-pi interactions (Hughes and Waters, 2006
). Such interactions are seen in the aromatic cage of the SMN tudor domain that likely interacts with the methylated tail of the SmD splicing factor (Sprangers et al., 2003
). Thus, modification of arginine residues in proteins can readily modulate their binding interactions and thus can regulate their physiological functions.
Three distinct types of methylated arginine residues occur in mammalian cells. The most prevalent is omega-NG
-dimethylarginine (Paik and Kim, 1980). Here, two methyl groups are placed on one of the terminal nitrogen atoms of the guanidino group; this derivative is commonly referred to as asymmetric dimethylarginine (ADMA) (). Two other derivatives occur at levels of about 20% to 50% that of ADMA. These include the symmetric dimethylated derivative, where one methyl group is placed on each of the terminal guanidino nitrogens (omega-NG
-dimethylarginine; SDMA) and the monomethylated derivative with a single methyl group on the terminal nitrogen atom (omega-NG
-monomethylarginine; MMA). These three derivatives are present on a multitude of distinct protein species in the cytoplasm, nucleus, and organelles of mammalian cells (Bedford and Richard, 2005
). Methylated arginine residues in proteins are often flanked by one or more glycine residues (Gary and Clarke, 1998
), but there are many exceptions to this general rule.
Types of methylation on arginine residues
The formation of MMA, ADMA, and SDMA in mammalian cells is performed by a sequence-related family of catalytic subunits of protein arginine methyltransferases termed PRMTs (). The exact number of genes encoding these catalytic subunits is under current investigation; six genes are known to encode enzymes with well-characterized activities (PRMT1, 3, 4 (CARM1), 5, 6, and 8), and another three genes encode sequence-related proteins with possible or probable methyltransferase activities (PRMT2, 7, 9 (4q31)) (Bedford, 2007
). Each PRMT species harbors the characteristic motifs of seven-beta strand methyltransferases (Katz et al., 2003
), as well as additional “double E” and “THW” sequence motifs particular to the PRMT subfamily (Cheng et al., 2005
). It has also been proposed that the FBXO11 and FBXO10 proteins, which do not harbor these signature motifs, represent a second family of protein arginine methyltransferases; however these activities require validation (Cook et al., 2006
; Krause et al., 2007
). The gene products with well-characterized activities, generally purified as fusion proteins, catalyze MMA formation; PRMT1, 3, 4 (CARM1), 6 and 8 additionally catalyze ADMA formation, whereas PRMT5 additionally catalyzes SDMA formation. Enzymes that form ADMA are designated “type I”; those that form SDMA are designated “type II” (Gary and Clarke, 1998
). To date, no enzyme has been found that forms both ADMA and SDMA derivatives.
The protein arginine methyltransferase family
A clear understanding of how PRMT catalytic polypeptides can be assembled into active complexes with other protein subunits in cells is needed. Although PRMT1, 3, 4(CARM1), 5, 6, and 8 are active methyltransferases in the absence of other polypeptide species in vitro, they might also bind additional partners for their functions in vivo. It now appears that interactions between PRMTs and their binding partners (and presumed regulatory subunits) can be transient or permanent (). It is possible that the species with poorly defined activities require the presence of other subunits for catalytic activity.
Interacting Protein Partners of Mammalian PRMTs
An important question under current investigation is whether protein arginine demethylation reactions occur to reverse the effects of the modifications. Initial studies indicated that methyl groups were stable on arginine residues. The apparent absence of protein arginine demethylases suggested that the only way to reverse the effects of the modification would be to degrade the protein to its component amino acids and then make a new unmodified version by protein synthesis. However, two types of enzymes that can remove methyl groups from arginine residues in proteins were recently identified. MMA residues in proteins can be deiminated to citrulline residues by the PAD4 peptidylarginine deiminase (Thompson and Fast, 2006
). It is unknown whether the citrulline residue might be converted to an arginine residue to complete the demethylation process; however it has been suggested that this enzyme is unlikely to play a physiological demethylation role. Indeed, peptides containing MMA are more slowly deiminated than those containing arginine residues and those containing ADMA are not deiminated at all; thus methylation might in fact inhibit a reaction that normally converts arginine residues to citrulline residues (Raijmakers et al., 2007
). Further work will be needed to assess the role of these enzymes in demethylation. Additionally, recent work has suggested that a second type of enzyme, the Jumonji domain-containing proteins, which was originally identified as a family of lysine demethylases, can also demethylate arginine residues. Indeed, JMJD6 can directly regenerate arginine residues from methylated histone species (Chang et al., 2007
). It will be important to determine if there are additional enzymes that catalyze similar reactions, and to assess the biological significance of each of these demethylation pathways in regulating protein arginine methylation.