Detection of a surface-exposed PEST like sequence in the metabotropic glutamate receptor mGluR1

Contact: [email protected] PHD protein secondary structure analysis software is a neural-network based method, rating at an expected average accuracy ∼72% for the three states helix, strand and loop when using multiple alignment input (Rost and Sander, 1993a,b, 1994). Using the PHD also allows the prediction of solvent accessibility (i.e. surface exposure) of individual amino-acid residues of a protein. PEST sequences are defined as hydrophilic stretches of polypeptide sequences rich in proline, glutamic acid, serine and threonine, and flanked by positively charged residues and are an important feature of eukaryotic proteins with intracellular half-lives of less than 2 h (Rogers et al., 1986; Rechsteiner et al., 1987; Rechsteiner and Rogers, 1996). The data accumulated since 1986 indicate strongly that PEST sequences serve as signals for proteolysis and their presence correlates well with ubiquitination, fast endocytosis and rapid protein degradation (by both proteasome and lysosomal proteases) but not with protein compartmentation or functional activity (Rechsteiner and Rogers, 1996, and references therein). Presence of PEST motifs do not necessarily lead to constitutive degradation of the protein, unless these sequences are surface exposed and not masked by the interacting proteins. If no structural information is available for a protein under study it is therefore necessary to also check for the surface accessibility of the predicted PEST sequences. The metabotropic glutamate receptors belong to the class of seven transmembrane receptors, which are coupled to G-proteins and mediate intracellular signal transduction. Unusually long C-terminus of the mGluR1α receptor appears to contain various sorting motifs which are critical for correct sorting and targeting of the receptor in neurones and their retention in the plasma membrane (Ciruela et al., 1999a). Recently a family of Homer proteins have been identified among the genes which are induced in neurones of the hippocampus and cortex by excitatory synaptic activity and during development Fig. 1. PHD and PESTfind analysis of the mGluR1α sequence. The rat metabotropic glutamate receptor sequence (SWISS-PROT Acc.No.: P23385) was submitted to the PredictProtein server available at the http://dodo.cpmc.columbia.edu (‘PHD’) and to the EMBnet Austria server at the http://emb1.bcc.univie.ac.at/embnet/ tools (‘PESTfind’). Amino acid sequence of the C-terminus of the mGluR1α (AA). PHDacc denotes the predicted relative solvent accessibility of amino acid residues (b—buried, e—surface exposed). PEST indicates the predicted PEST sequences (+). The boxed sequence indicate a Homer-binding motif reported earlier (Tu et al., 1998). (Brakeman et al., 1997; Kato et al., 1997). The Homer proteins are produced from three genes and a number of various splicing forms of the Homer proteins have been recently described (Soloviev et al., 2000). Homer proteins bind to the Pro.Pro.x.x.Phe.Arg motif found in the C-terminus of mGluR1α receptor and in other proteins. Recently an evidence for the role of Homer proteins in cell surface targeting and anchoring of mGluR1 receptors have been provided (Ciruela et al., 1999b, 2000). Moreover, Ciruela et al. have also reported a significant increase in the amount of total mGluR1α protein detected upon its co-expression with the Homer-1a protein in HEK-293 cells. The mechanism responsible for the increased stability of the mGluR1α proteins is not yet clear. Transcriptional and/or translational control by Homer proteins could be responsible for such changes in the c © Oxford University Press 2000 837