The GTPase dynamin regulates endocytic vesicle budding from the plasma membrane, but the molecular mechanisms involved remain incompletely understood. We report that dynamin, which interacts with NO synthase, is S-nitrosylated at a single cysteine residue (C607) after stimulation of the ␤2 adrenergic receptor. S-nitrosylation increases dynamin self-assembly and GTPase activity and facilitates its redistribution to the membrane. A mutant protein bearing a C607A substitution does not self-assemble properly or increase its enzymatic activity in response to NO. In NO-generating cells, expression of dynamin C607A, like the GTPase-deficient dominant-negative K44A dynamin, inhibits both ␤2 adrenergic receptor internalization and bacterial invasion. Furthermore, exogenous or endogenously produced NO enhances internalization of both ␤2 adrenergic and epidermal growth factor receptors. Thus, NO regulates endocytic vesicle budding by S-nitrosylation of dynamin. Collectively, our data suggest a general NO-dependent mechanism by which the trafficking of receptors may be regulated and raise the idea that pathogenic microbes and viruses may induce S-nitrosylation of dynamin to facilitate cellular entry.receptor ͉ infection ͉ protein-protein interaction T he response of cells to their environment is partly determined by the complement of receptors expressed on the plasma membrane. Surface receptor expression is a function of a dynamic equilibrium between vesicle-confined receptor endocytosis (internalization) and receptor exocytosis (recycling and synthesis). The large GTPase dynamin plays a central role in the endocytotic budding of vesicles from the plasma membrane (1). Dynamin is thought to act as either a mechanochemical enzyme that executes vesiculation (2, 3) or, like other GTPases, to control downstream effector(s) that, in turn, mediate the vesicle fission (4). In particular, dynamin self-assembles to form a collar at the neck of invaginating pits and hydrolyzes GTP in the act of fission (5). Although dynamin activity can be regulated by phosphorylation (6-8) and by interactions with lipids (9) and partner proteins (10-12), the mechanisms of self-assembly and regulated GTP hydrolysis remain poorly understood.Dynamin binds endothelial NO synthase (eNOS) (13), and emerging evidence suggests that eNOS may regulate vesicle trafficking (14, 15). It has been proposed that one key determinant of specificity in NO signaling is the interaction between NOSs and proteins that are targets of S-nitrosylation (16). We have therefore tested the hypothesis that NOS and its product NO regulate vesicular trafficking through S-nitrosylation of dynamin. ResultsActivation of ␤ 2 adrenergic receptors (␤ 2 AR) with isoproterenol (ISO) initiates dynamin-dependent receptor internalization (17), which we used as a measure of vesicle trafficking from the plasma membrane. Although NO is ubiquitous in vivo, most studies examining receptor internalization have been carried out in model cell lines that do not exhibit measurable NOS activity [e.g., hu...

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