Abstract
The huntingtin-interacting protein family members (Hip1 and Hip1R in mammals and Sla2p in yeast) link clathrin-mediated membrane traffic to actin cytoskeleton dynamics. Genetic data in yeast have implicated the light chain subunit of clathrin in regulating this link. To test this hypothesis, the biophysical properties of mammalian Hip1 and Hip1R and their interaction with clathrin light chain and actin were analyzed. The coiled-coil domains (clathrin light chain-binding) of Hip1 and Hip1R were found to be stable homodimers with no propensity to heterodimerize in vitro. Homodimers were also predominant in vivo, accounting for cellular segregation of Hip1 and Hip1R functions. Coiled-coil domains of Hip1 and Hip1R differed in their stability and flexibility, correlating with slightly different affinities for clathrin light chain and more markedly with effects of clathrin light chain binding on Hip protein-actin interactions. Clathrin light chain binding induced a compact conformation of both Hip1 and Hip1R and significantly reduced actin binding by their THATCH domains. Thus, clathrin is a negative regulator of Hip-actin interactions. These observations necessarily change models proposed for Hip protein function.
Highlights
Androgen receptor activation, and the pathogenesis of Huntington disease [5,6,7,8,9]
Overexpression of the Hip proteinbinding region of clathrin light chain causes an alteration in actin cytoskeleton structure, generating short actin protrusions tipped with cortactin [4]
Hip1 and Hip1R Are Stable Homodimers—Homodimers of Hip1R have an elongated dumbbell shape, with the N- and C-terminal globular domains in variable positions [32]. This implies the primary dimerization determinants are within the coiled-coil domain
Summary
Expression Constructs and Peptides—Separate domains of either human Hip or mouse Hip1R were cloned. Full-length Hip or Hip1R were amplified by PCR with primers containing either the polyhistidine (His) tag or hemagglutinin (HA) tag sequence and cloned into the pCDNA3.1 plasmid (Invitrogen). Clathrin light chain LCb was expressed from a previously described construct [27]. Cortactin Src homology 3 domain fused with glutathione S-transferase (GST) was expressed and purified as previously described [17]. A peptide representing the consensus sequence of clathrin light chains (EEDPAAAFLAQQESEIAGIEND) was synthesized commercially. Control peptides of the same length were either the clathrin light chain peptide with three previously described point mutations (underlined) that abrogate Hip1R binding [4] (EEVPAAAFLAQQESEAAGIAND) or unrelated peptide (FINKPETGAVELESPFILLADKKI) derived from the bacterial protein GroEL
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