Role of the basic, proline-rich region of dynamin in Src homology 3 domain binding and endocytosis.

The GTPase dynamin has been implicated in the regulation of the scission of coated and noncoated pits during the early stages of endocytosis. Various macromolecules including microtubules, acidic phospholipids, and Src homology 3 (SH3) domains have been shown to interact with the basic, proline-rich region of dynamin and act as effectors of its GTPase activity. The interaction of dynamin with SH3 domain-containing proteins is of particular interest since SH3 domains are known to mediate protein-protein interactions in signal transducing complexes. In this study, we have systematically defined three distinct SH3 binding regions within the dynamin proline-rich C terminus. These binding regions conform to either the Class I or II SH3 binding consensus sequence, and their location coincides with a region previously shown to be important in the colocalization of dynamin with clathrin-coated pits. Two of these SH3 binding regions are well conserved among four dynamin isoforms, and we show that the overall binding pattern for SH3 domains is comparable among the isoforms. We also demonstrate that neither transferrin nor platelet-derived growth factor receptor uptake is restored upon removal of the basic, proline-rich region in a dominant negative dynamin GTP binding mutant. Together with earlier evidence from our laboratory, these findings suggest that SH3 domains may serve to target dynamin to coated pits and are not the direct targets of dominant inhibitory mutants of dynamin.

A binding site for SH3 domains targets dynamin to coated pits.

Dynamin is a GTPase that plays a critical role in the very early stages of endocytosis, regulating the scission of clathrin-coated and non-clathrin-coated pits from the plasma membrane. While the ligands through which dynamin exerts its in vivo effects are unknown, dynamin exhibits in vitro binding to several proteins containing Src homology 3 (SH3) domains, as well as to microtubules and anionic phospholipids, via a basic, proline-rich C-terminal domain. To begin to identify the in vivo binding partners of dynamin, we have examined by immunofluorescence the association of mutant and wild-type forms of dynamin with plasma membranes prepared by sonication of transiently transfected cells. Wild-type dynamin was found almost exclusively in association with clathrin-containing domains. Binding to these regions was abolished by removal of a nine-amino acid sequence within the C-terminal domain encoding a candidate SH3 domain binding site. Binding did not require clathrin and resisted extraction at both high and low ionic strength, consistent with an interaction with an SH3 domain. Surprisingly, we also find that dynamin contains multiple regions involved in binding to nonclathrin-containing domains, including a 13-amino acid sequence directly upstream of the C-terminal domain. These observations suggest that a protein containing an SH3 domain is involved in recruiting dynamin to coated pits and provide the first evidence for a biological role for SH3 domains in dynamin function.

Molecular analysis of a cytoplasmic dynein light intermediate chain reveals homology to a family of ATPases.

Cytoplasmic dynein is a multi-subunit complex involved in retrograde organelle transport and some aspects of mitosis. In previous work we have cloned and sequenced cDNAs encoding the rat cytoplasmic dynein heavy and intermediate chains. Here we report the cloning of the remaining class of cytoplasmic dynein subunits, which we refer to as the light intermediate chains (LICs: 53-59 kDa). Four LIC electrophoretic bands were resolved in purified bovine cytoplasmic dynein preparations by one-dimensional gel electrophoresis. These four bands were simplified to two bands (LIC53/55 and LIC57/59) by alkaline phosphatase treatment. N-terminal amino acid sequence was obtained from a total of 11 proteolytic peptides generated from both LIC53/55 and LIC57/59. Overlapping cDNA clones encoding LIC53/55 were isolated by oligonucleotide screening using probes based on the LIC53/55 peptide sequence. The cDNA sequence contained a 497 codon open reading frame encoding a polypeptide with a molecular mass of approximately 55 kDa. Each of the LIC53/55 peptides was found within the deduced amino acid sequence, as well as four of the LIC57/59 peptides. Analysis of the LIC53/55 primary sequence revealed homology with the ABC transporter family of ATPases in the region surrounding the P-loop sequence element. Together these data identify the LICs as a novel family of dynein subunits with potential ATPase activity. They also reveal that the complexity of the LICs is due to both post-translational modification and the existence of at least two LIC polypeptides for which we propose the names LIC-1a and LIC-2.

Microtubules and Src homology 3 domains stimulate the dynamin GTPase via its C-terminal domain.

Dynamin is a 100-kDa GTPase that plays a critical role in the initial stages of endocytosis. Dynamin binds to microtubules, which potently stimulate its GTPase activity. Binding to Src homology 3 (SH3) domains of proteins involved in signal transduction has also recently been reported. In the present study, the protein was digested with a variety of proteases to define its functional domains. Limited digestion with papain split the protein into an approximately 7- to 9-kDa microtubule-binding fragment and a 90-kDa nonbinding fragment. Immunoblotting with an antibody to the C-terminal 20 amino acids of rat dynamin showed the small fragment to derive from the C-terminal end of the polypeptide. Microtubule-activated GTPase activity, but not basal GTPase activity, was abolished by papain digestion, identifying the basic, proline-rich C-terminal region of dynamin as an important regulatory site. Bacterially expressed growth factor receptor-bound protein 2 (GRB2) and the SH3 domain of c-Src were also found to stimulate GTPase activity, although to a lesser extent than microtubules. Stimulation of GTPase activity by the recombinant proteins was similarly abolished by papain digestion. These results identify the basic, proline-rich C-terminal region of dynamin as the binding site for both microtubules and SH3 domains and demonstrate an allosteric interaction between this region of the molecule and the N-terminal GTPase domain.

Effects of mutant rat dynamin on endocytosis.

Dynamin is a 100-kD microtubule-activated GTPase. Recent evidence has revealed a high degree of sequence homology with the product of the Drosophila gene shibire, mutations in which block the recycling of synaptic vesicles and, more generally, the formation of coated and non-coated vesicles at the plasma membrane. We have now transfected cultured mammalian COS-7 cells with both wild-type and mutant dynamin cDNAs. Point mutations in the GTP-binding consensus sequence elements of dynamin equivalent to dominant negative mutations in ras, and an NH2-terminal deletion of the entire GTP-binding domain of dynamin, block transferrin uptake and alter the distribution of clathrin heavy chain and alpha-, but not gamma-, adaptin. COOH-terminal deletions reverse these effects, identifying this portion of dynamin as a site of interaction with other components of the endocytic pathway. Over-expression of neither wild-type nor mutant forms of dynamin affected the distribution of microtubules. These results demonstrate a specific role for dynamin and for GTP in the initial stages of receptor-mediated endocytosis.

Dynamin, a GTPase involved in the initial stages of endocytosis.

Dynamin is a high molecular mass (100 kDa) GTPase which binds to and co-purifies with microtubules. Molecular cloning of rat brain dynamin has revealed the three well-established consensus sequence elements for GTP binding within the N-terminal third of the protein, as well as sequence similarity within this region to the interferon-inducible antiviral Mx proteins, the product of the yeast membrane sorting gene VPS1, and the product of the yeast mitochondrial replication gene MGM1. More extensive sequence similarity between rat dynamin and the product of the Drosophila gene shibire, which is involved in endocytosis, has also been found. In in vitro assays microtubules strongly stimulate the dynamin GTPase. This effect can be reversed by removal of the dynamin C-terminus using papain, which abolishes microtubule binding. Overexpression of mutant forms of dynamin in vivo using Cos-7 cells inhibits transferrin uptake and alters the distribution of clathrin and of alpha-adaptin, but not gamma-adaptin. Deletion of the C-terminus of mutant forms of dynamin abolishes these effects. Together these results suggest a critical role for dynamin in the early stages of endocytosis. It is uncertain whether microtubules interact with dynamin in vivo or whether the in vitro effects of microtubules mimic the effects of other regulatory elements in vivo.