Elaboration of the Homer1 recognition landscape reveals incomplete divergence of paralogous EVH1 domains.

Short sequences that mediate interactions with modular binding domains are ubiquitous throughout eukaryotic proteomes. Networks of short linear motifs (SLiMs) and their corresponding binding domains orchestrate many cellular processes, and the low mutational barrier to evolving novel interactions provides a way for biological systems to rapidly sample selectable phenotypes. Mapping SLiM binding specificity and the rules that govern SLiM evolution is fundamental to uncovering the pathways regulated by these networks and developing the tools to manipulate them. We used high-throughput screening of the human proteome to identify sequences that bind to the Enabled/VASP homology 1 (EVH1) domain of the postsynaptic density scaffolding protein Homer1. This expanded our understanding of the determinants of Homer EVH1 binding preferences and defined a new motif that can facilitate the discovery of additional Homer-mediated interactions. Interestingly, the Homer1 EVH1 domain preferentially binds to sequences containing an N-terminally overlapping motif that is bound by the paralogous family of Ena/VASP actin polymerases, and many of these sequences can bind to EVH1 domains from both protein families. We provide evidence from orthologous EVH1 domains in pre-metazoan organisms that the overlap in human Ena/VASP and Homer binding preferences corresponds to an incomplete divergence from a common Ena/VASP ancestor. Given this overlap in binding profiles, promiscuous sequences that can be recognized by both families either achieve specificity through extrinsic regulatory strategies or may provide functional benefits via multi-specificity. This may explain why these paralogs incompletely diverged despite the accessibility of further diverged isoforms.

The physiological role of Homer2a and its novel short isoform, Homer2e, in NMDA receptor-mediated apoptosis in cerebellar granule cells.

Homer is a postsynaptic scaffold protein, which has long and short isoforms. The long form of Homer consists of an N-terminal target-binding domain and a C-terminal multimerization domain, linking multiple proteins within a complex. The short form of Homer only has the N-terminal domain and likely acts as a dominant negative regulator. Homer2a, one of the long form isoforms of the Homer family, expresses with a transient peak in the early postnatal stage of mouse cerebellar granule cells (CGCs); however, the functions of Homer2a in CGCs are not fully understood yet. In this study, we investigated the physiological roles of Homer2a in CGCs using recombinant adenovirus vectors. Overexpression of the Homer2a N-terminal domain construct, which was made structurally reminiscent with Homer1a, altered NMDAR1 localization, decreased NMDA currents, and promoted the survival of CGCs. These results suggest that the Homer2a N-terminal domain acts as a dominant negative protein to attenuate NMDAR-mediated excitotoxicity. Moreover, we identified a novel short form N-terminal domain-containing Homer2, named Homer2e, which was induced by apoptotic stimulation such as ischemic brain injury. Our study suggests that the long and short forms of Homer2 are involved in apoptosis of CGCs.

Interaction of the Homer1 EVH1 domain and skeletal muscle ryanodine receptor.

The skeletal muscle ryanodine receptor (RyR1) proteins are intracellular calcium (Ca2+) release channels on the membrane of the sarcoplasmic reticulum (SR) and required for skeletal muscle excitation-contraction coupling. Homer (Vesl) is a family of scaffolding proteins that modulate target proteins including RyRs (ryanodine receptors), mGluRs (group 1 metabotropic glutamate receptors) and IP3Rs (inositol-1,4,5-trisphosphate receptors) through a conserved EVH1 (Ena/VASP homology 1) domain. Here, we examined the interaction between Homer1 EVH1 domain and RyR1 by co-immunoprecipitation, continuous sucrose density-gradient centrifugation, and bio-layer interferometry binding assay at different Ca2+ concentrations. Our results show that there exists a high-affinity binding between the Homer1 EVH1 domain and RyR1, especially at 1 mM of Ca2+. Based on our data and the known structures of Homer1 EVH1 domain and RyR1, we found two consensus proline-rich sequences in the structure of RyR1, PPHHF and FLPPP, and proposed two corresponding binding models to show mechanisms of recognition different from those used by other proline-rich motifs. The side proline residues of two proline-rich motifs from RyR1 are away from the hydrophobic surface of Homer1 EVH1, rather than buried in this hydrophobic surface. Our results provide evidence that Homer1 regulates RyR1 by direct interaction.

Homer Tetramer Promotes Actin Bundling Activity of Drebrin.

Drebrin is an actin bundling protein that plays critical roles in synaptic spine development and plasticity. Homer, one of the most abundant scaffolding proteins in postsynaptic density, interacts with Drebrin's C-terminal PPXXF motifs using its Ena/VASP homology 1 (EVH1) domain. However, the molecular mechanism and biological function of this interaction remain unclear. Here we show that Homer specifically binds to the first but not the second PPXXF motif in Drebrin. The crystal structure of Drebrin-Homer binding motif 1 in complex with Homer EVH1 reveals a consensus Homer EVH1 binding motif. Homer tetramer promotes actin bundling activity of Drebrin in vitro and stimulates Drebrin-induced filopodia formation and elongation in cells. We further show that monomeric Homer1a antagonizes Homer1b in promoting Drebrin-stimulated actin bundling. Our study suggests a potential regulatory role of Homer1 in modulating excitatory synaptic spine homeostatic scaling via binding to Drebrin.

Whole exome sequencing identified a second pathogenic variant in HOMER2 for autosomal dominant non-syndromic deafness.

Hearing loss is one of the most common sensory disorders worldwide, and about half of all occurrences are attributable to genetic factors. Here, we have identified a novel pathogenic variant in HOMER2 in a Chinese family with autosomal dominant, non-syndromic hearing loss. This is the second family reported globally with hearing loss caused by a variant in HOMER2. The pathogenic variant c.840_841insC in HOMER2 (NM_199330), segregating with the hearing-loss phenotype in the family, leads to a premature stop codon producing a truncated protein. The coiled-coil domain in the C-terminal of HOMER2 protein is essential for protein multimerization and HOMER2-CDC42 interaction. We compared the phenotypes in the two families and found that hearing impairment in this Chinese family was more severe. Furthermore, we found that the ability of this insertion mutant type HOMER2 (HOMER2MU ) to multimerize decreased more significantly than wild-type HOMER2 (HOMER2WT ) and the reported c.554G>C (NM_004839) mutant HOMER2. HOMER2MU protein tended to be distributed in a diffuse manner, whereas HOMER2WT and the reported mutant HOMER2 tended to cluster together. Our research provides a validating second family for variants in HOMER2 causing non-syndromic sensorineural hearing loss. HOMER2 homo-/hetero-multimerization might be the first step in exerting its normal function.