Myosin-X recruits lamellipodin to filopodia tips.

Myosin-X (MYO10), a molecular motor localizing to filopodia, is thought to transport various cargo to filopodia tips, modulating filopodia function. However, only a few MYO10 cargoes have been described. Here, using GFP-Trap and BioID approaches combined with mass spectrometry, we identified lamellipodin (RAPH1) as a novel MYO10 cargo. We report that the FERM domain of MYO10 is required for RAPH1 localization and accumulation at filopodia tips. Previous studies have mapped the RAPH1 interaction domain for adhesome components to its talin-binding and Ras-association domains. Surprisingly, we find that the RAPH1 MYO10-binding site is not within these domains. Instead, it comprises a conserved helix located just after the RAPH1 pleckstrin homology domain with previously unknown functions. Functionally, RAPH1 supports MYO10 filopodia formation and stability but is not required to activate integrins at filopodia tips. Taken together, our data indicate a feed-forward mechanism whereby MYO10 filopodia are positively regulated by MYO10-mediated transport of RAPH1 to the filopodium tip.

The C5 Variant of the Butyrylcholinesterase Tetramer Includes a Noncovalently Bound 60 kDa Lamellipodin Fragment.

Humans with the C5 genetic variant of butyrylcholinesterase (BChE) have 30-200% higher plasma BChE activity, low body weight, and shorter duration of action of the muscle relaxant succinylcholine. The C5 variant has an extra, slow-moving band of BChE activity on native polyacrylamide gel electrophoresis. This band is about 60 kDa larger than wild-type BChE. Umbilical cord BChE in 100% of newborn babies has a C5-like band. Our goal was to identify the unknown, 60 kDa protein in C5. Both wild-type and C5 BChE are under the genetic control of two independent loci, the BCHE gene on Chr 3q26.1 and the RAPH1 (lamellipodin) gene on Chr 2q33. Wild-type BChE tetramers are assembled around a 3 kDa polyproline peptide from lamellipodin. Western blot of boiled C5 and cord BChE showed a positive response with an antibody to the C-terminus of lamellipodin. The C-terminal exon of lamellipodin is about 60 kDa including an N-terminal polyproline. We propose that the unknown protein in C5 and cord BChE is encoded by the last exon of the RAPH1 gene. In 90% of the population, the 60 kDa fragment is shortened to 3 kDa during maturation to adulthood, leaving only 10% of adults with C5 BChE.

Association between RAPH1 Gene Haplotypes and CHE2 Locus Phenotypes.

The human butyrylcholinesterase (BChE) is a serum esterase that has been associated with body mass index (BMI) and obesity. Its activity is conditioned by alleles of BCHE gene and the CHE2 locus that codifies an unknown BChE-binding protein (C5 complex). The hypothesis that the CHE2 locus is the RAPH1 gene, which encodes lamellipodin (Lpd), was raised in a study that observed Lpd peptides released from denatured BChE tetramers. The aim of this study was to test this hypothesis by evaluating SNPs of RAPH1 gene (rs2246118:C > T, rs3814365:A > G and rs2465520:C > T) in 34 CHE2 C5+ and 92 CHE2 C5- individuals, corresponding to the presence and absence of C5 complex. The results showed association of two haplotypes (CAC and TGC) with CHE2 C5+ phenotype. RAPH1 haplotypes was also associated with intense (TGC) and faint (CAC) CHE2 C5+ phenotypes. BChE activity was higher in intense CHE2 C5+ than faint CHE2 C5+ phenotype. Our results corroborate the hypothesis that the RAPH1 gene is the CHE2 locus and suggest that the variable expressivity of the CHE2 C5+ phenotypes is, at least in part, due to its genetic heterogeneity, which is leading to increased BChE activity only in individuals with intense CHE2 C5+ phenotype.