Cloning and characterization of intermediate Homer1E of human skeletal muscle

HOMER proteins are scaffolding proteins critically involved in intracellular signaling, calcium homeostasis, receptor trafficking and synaptic plasticity. Three human HOMER genes (HOMER1, HOMER2, HOMER3) are expressed in both neurons and skeletal muscle fibers. HOMER1 long isoforms multimerize via their carboxy-terminal coiled-coil domain, forming signaling clusters with postsynaptic density proteins. Despite growing evidence of HOMER1 relevance in skeletal muscle physiology, molecular data on human muscle remain scarce. This study investigates the expression of alternatively spliced HOMER1 transcripts in human skeletal muscle, with focus on a previously uncharacterized intermediate isoform, HOMER1E. Human skeletal muscle biopsies (Soleus and Vastus Lateralis) and cerebellum were analyzed by RT-PCR and droplet digital PCR (ddPCR) to quantify HOMER1 transcript variants. HOMER1E cDNA was cloned and expressed in HEK293 cells alongside full-length HOMER1. Protein stability was assessed using cycloheximide chase assays. Degradation pathways were investigated with MG-132 (proteasome inhibitor) and Bafilomycin A1 (autophagy inhibitor). Protein-protein interactions were evaluated by co-affinity purification and confocal immunofluorescence. Structural modeling employed AlphaFold-Multimer and DeepCoil predictions. Three HOMER1 transcripts (HOMER1, HOMER1H, HOMER1E) were detected in human skeletal muscle, with HOMER1E representing ~ 0.2% of total HOMER1 transcripts, as determined by ddPCR. HOMER1E encodes a 224 aa, 25.8 kDa protein which lacks exons 4-6 (including the autoinhibitory P-motif) but retains both N- and C-termini. In HEK293 cells, HOMER1E protein was highly unstable, primarily degraded via autophagy, while co-expression with HOMER1 significantly stabilized it. Co-affinity purification and immunofluorescence confirmed direct HOMER1-HOMER1E interaction. Structural modeling predicted a coiled-coil-mediated antiparallel heterodimer interface. HOMER1E is a minor, unstable HOMER1 isoform in human skeletal muscle with no murine counterpart. Its interaction with full-length HOMER1 via the conserved coiled-coil domain, combined with its rapid autophagic turnover, suggests a regulatory role in modulating multimeric HOMER1 scaffolding under specific physiological or developmental conditions.