Clathrin hub expression dissociates the actin-binding protein Hip1R from coated pits and disrupts their alignment with the actin cytoskeleton.

The actin cytoskeleton has been implicated in the maintenance of discrete sites for clathrin-coated pit formation during receptor-mediated endocytosis in mammalian cells, and its function is intimately linked to the endocytic pathway in yeast. Here we demonstrate that staining for mammalian endocytic clathrin-coated pits using a monoclonal antibody against the AP2 adaptor complex revealed a linear pattern that correlates with the organization of the actin cytoskeleton. This vesicle organization was disrupted by treatment of cells with cytochalasin D, which disassembles actin, or with 2,3-butanedione monoxime, which prevents myosin association with actin. The linear AP2 staining pattern was also disrupted in HeLa cells that were induced to express the Hub fragment of the clathrin heavy chain, which acts as a dominant-negative inhibitor of receptor-mediated endocytosis by direct interference with clathrin function. Additionally, Hub expression caused the actin-binding protein Hip1R to dissociate from coated pits. These findings indicate that proper function of clathrin is required for coated pit alignment with the actin cytoskeleton and suggest that the clathrin-Hip1R interaction is involved in the cytoskeletal organization of coated pits.

Inhibition of human mast cell activation with the novel selective adenosine A(2B) receptor antagonist 3-isobutyl-8-pyrrolidinoxanthine (IPDX)(2).

The antiasthmatic drug enprofylline was the first known selective, though not potent, A(2B) antagonist. On the basis of structure-activity relationships (SARs) of xanthine derivatives, we designed a novel selective adenosine A(2B) receptor antagonist, 3-isobutyl-8-pyrrolidinoxanthine (IPDX), with potency greater than that of enprofylline. IPDX displaced [3H]ZM241385 ([3H]4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a]-[1,3,5]triazin-5-ylamino]ethyl)phenol) from human A(2B) adenosine receptors with a K(i) value of 470 +/- 2 nM and inhibited A(2B)-dependent cyclic AMP (cAMP) accumulation in human erythroleukemia (HEL) cells with a K(B) value of 625 +/- 71 nM. We found that IPDX was more selective than enprofylline toward human A(2B) receptors. It was 38-, 55-, and 82-fold more selective for human A(2B) than for human A(1) (K(i) value of 24 +/- 8 microM), human A(2A) (K(B) value of 36 +/- 8 microM), and human A(3) (K(i) value of 53 +/- 10 microM) adenosine receptors, respectively. IPDX inhibited NECA (5'-N-ethylcarboxamidoadenosine)-induced interleukin-8 secretion in human mast cells (HMC-1) with a potency close to that determined for A(2B)-mediated cAMP accumulation in HEL cells, thus confirming the role of A(2B) adenosine receptors in mediating human mast cell activation. Since adenosine triggers bronchoconstriction in asthmatic patients through human mast cell activation, IPDX may become a basis for the development of new antiasthmatic drugs with improved properties compared with those of enprofylline. Our data demonstrate that IPDX can be used as a tool to differentiate between A(2B) and other adenosine receptor-mediated responses.

The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro.

Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast. Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis. First, real-time analysis of Hip1R-YFP and DsRed-clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex. Second, at the ultrastructure level, immunogold labeling of 'unroofed' cells showed that Hip1R localizes to clathrin-coated pits. Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC. Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro. Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures. In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

Nodular amyloidosis: case report and literature review.

BACKGROUND: Amyloidosis refers to a group of depositional diseases that are classified into two main types: systemic and localized. Large nodules of localized cutaneous amyloidosis of the nasal ala and surrounding skin are rare and the treatment is often unsatisfactory. OBJECTIVE: We report a case of rapidly enlarging, localized, nodular cutaneous amyloidosis of the nose and the surrounding skin with a brief review of the current literature regarding treatment of this rare disease. CONCLUSION: Nodular amyloidosis can be treated successfully with cold steel excision in combination with carbon dioxide laser. Close followup of these patients is warranted, as nodular amyloidosis may be the precursor to systemic amyloidosis.

Cutaneous Acanthamoeba in a patient with AIDS: a case study with a review of new therapy; quiz 386.

GOAL: To describe the presenting signs of an Acanthamoeba infection. OBJECTIVES: Upon completion of this activity, dermatologists and general practitioners should be able to: 1. Discuss the clinical presentation of Acanthamoeba infection. 2. Describe the conditions that make a patient susceptible to Acanthamoeba. 3. Outline treatment options for Acanthamoeba infection. CME: This article has been peer reviewed and approved by Michael Fisher, MD, Professor of Medicine, Albert Einstein College of Medicine. REVIEW DATE: April 2001. This activity has been planned and implemented in accordance with the Essentials and Standards of the Accreditation Council for Continuing Medical Education through the joint sponsorship of Albert Einstein College of Medicine and Quadrant HealthCom, Inc. The Albert Einstein College of Medicine is accredited by the ACCME to provide continuing medical education for physicians. Albert Einstein College of Medicine designates this educational activity for a maximum of 1.0 hour in category 1 credit toward the AMA Physician's Recognition Award. Each physician should claim only those hours of credit that he/she actually spent in the educational activity. This activity has been planned and produced in accordance with ACCME Essentials.

HIP12 is a non-proapoptotic member of a gene family including HIP1, an interacting protein with huntingtin.

Huntingtin-interacting protein I (HIP1) is a membrane-associated protein that interacts with huntingtin, the protein altered in Huntington disease. HIP1 shows homology to Sla2p, a protein essential for the assembly and function of the cytoskeleton and endocytosis in Saccharomyces cerevisiae. We have determined that the HIP1 gene comprises 32 exons spanning approximately 215 kb of genomic DNA and gives rise to two alternate splice forms termed HIP1-1 and HIP1-2. Additionally, we have identified a novel protein termed HIP12 with significant sequence and biochemical similarities to HIP1 and high sequence similarity to Sla2p. HIP12 differs from HIP1 in its pattern of expression both at the mRNA and protein level. However, HIP1 and HIP12 are both found within the brain and show a similar subcellular distribution pattern. In contrast to HIP1, which is toxic in cell culture, HIP12 does not confer toxicity in the same assay systems. Interestingly, HIP12 does not interact with huntingtin but can interact with HIP1. suggesting a potential interaction in vivo that may influence the function of each respective protein.

Cyclic AMP and protein kinase A stimulate Cdc42: role of A(2) adenosine receptors in human mast cells.

The functional activity of Cdc42 is known to be regulated by proteins that control its GDP/GTP-bound state. However, there is still limited information on how Cdc42 is controlled by G-protein-coupled receptors. Adenosine receptors belong to the G-protein-coupled receptor family of cell surface receptors. Human HMC-1 mast cells express the high-affinity A(2A) and the low-affinity A(2B) subtypes of adenosine receptors known to increase intracellular cAMP levels. We found that both subtypes of A(2) adenosine receptors activate Cdc42 in HMC-1 cells. Furthermore, stimulation of adenylate cyclase with forskolin, or loading of HMC-1 with the cell-permeable cAMP analog 8-Br-cAMP, activated Cdc42. Stimulation of Cdc42 by cAMP was also observed in CHO-K1 and COS-7 cells. Protein kinase A (PKA)-mediated phosphorylation is likely involved in cAMP-dependent Cdc42 activation, because transient expression of the PKA catalytic subunit in COS-7 cells activated Cdc42. Inhibition of protein phosphatases 1 and 2A with calyculin A potentiated the effects of 5'-N-ethylcarboxamidoadenosine and 8-Br-cAMP, whereas the selective PKA inhibitor H-89 reversed the activation of Cdc42. We demonstrated that Cdc42 is a poor substrate for PKA phosphorylation in vitro and in intact cells. Our data suggest that PKA does not phosphorylate Cdc42 directly. Instead, the proteins that modulate the GDP/GTP-bound state of Cdc42 may be the primary targets of PKA phosphorylation.

Association of mouse actin-binding protein 1 (mAbp1/SH3P7), an Src kinase target, with dynamic regions of the cortical actin cytoskeleton in response to Rac1 activation.

Yeast Abp1p is a cortical actin cytoskeleton protein implicated in cytoskeletal regulation, endocytosis, and cAMP-signaling. We have identified a gene encoding a mouse homologue of Abp1p, and it is identical to SH3P7, a protein shown recently to be a target of Src tyrosine kinases. Yeast and mouse Abp1p display the same domain structure including an N-terminal actin-depolymerizing factor homology domain and a C-terminal Src homology 3 domain. Using two independent actin-binding domains, mAbp1 binds to actin filaments with a 1:5 saturation stoichiometry. In stationary cells, mAbp1 colocalizes with cortical F-actin in fibroblast protrusions that represent sites of cellular growth. mAbp1 appears at the actin-rich leading edge of migrating cells. Growth factors cause mAbp1 to rapidly accumulate in lamellipodia. This response can be mimicked by expression of dominant-positive Rac1. mAbp1 recruitment appears to be dependent on de novo actin polymerization and occurs specifically at sites enriched for the Arp2/3 complex. mAbp1 is a newly identified cytoskeletal protein in mice and may serve as a signal-responsive link between the dynamic cortical actin cytoskeleton and regions of membrane dynamics.

An actin-binding protein of the Sla2/Huntingtin interacting protein 1 family is a novel component of clathrin-coated pits and vesicles.

The actin cytoskeleton has been implicated in endocytosis, yet few molecules that link these systems have been identified. Here, we have cloned and characterized mHip1R, a protein that is closely related to huntingtin interacting protein 1 (Hip1). These two proteins are mammalian homologues of Sla2p, an actin binding protein important for actin organization and endocytosis in yeast. Sequence alignments and secondary structure predictions verified that mHip1R belongs to the Sla2 protein family. Thus, mHip1R contains an NH(2)-terminal domain homologous to that implicated in Sla2p's endocytic function, three predicted coiled-coils, a leucine zipper, and a talin-like actin-binding domain at the COOH terminus. The talin-like domain of mHip1R binds to F-actin in vitro and colocalizes with F-actin in vivo, indicating that this activity has been conserved from yeast to mammals. mHip1R shows a punctate immunolocalization and is enriched at the cell cortex and in the perinuclear region. We concluded that the cortical localization represents endocytic compartments, because mHip1R colocalizes with clathrin, AP-2, and endocytosed transferrin, and because mHip1R fractionates biochemically with clathrin-coated vesicles. Time-lapse video microscopy of mHip1R-green fluorescence protein (GFP) revealed a blinking behavior similar to that reported for GFP-clathrin, and an actin-dependent inward movement of punctate structures from the cell periphery. These data show that mHip1R is a component of clathrin-coated pits and vesicles and suggest that it might link the endocytic machinery to the actin cytoskeleton.

Role of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinase kinase in adenosine A2B receptor-mediated interleukin-8 production in human mast cells.

The endogenous nucleoside adenosine is thought to play a role in the pathophysiology of asthma by stimulating mast cells. We previously showed that the human mast cell line HMC-1 expresses A2A and A2B receptors, and that both receptors activate adenylate cyclase via Gs-protein but that only A2B receptors are also coupled to phospholipase C via Gq proteins. Stimulation of A2B but not A2A receptors induced production of interleukin-8 (IL-8) from HMC-1 cells. The mechanism by which adenosine promotes IL-8 synthesis has not been defined. In this study, we tested the hypothesis that mitogen-activated protein kinase (MAPK) signaling pathways are involved in this process. Stimulation of HMC-1 with the stable adenosine analog NECA (5'-N-ethylcarboxamidoadenosine) activated p21(ras) and both p42 and p44 isoforms of extracellular signal-regulated kinase (ERK). NECA (10 microM) induced a 1.9 +/- 0. 06-fold increase in ERK activity, whereas 10 microM of the selective A2A agonist CGS 21680 (4-((N-ethyl-5'-carbamoyladenos-2-yl)-aminoethyl)-phenylpropionic acid) had no effect. NECA, in parallel with the activation of ERK, also stimulated the p46 isoform of c-Jun N-terminal kinase (MEK) and p38 MAPK. Furthermore, the selective MAPK/ERK kinase 1 inhibitor PD 98059 (2'-amino-3'-methoxyflavone), and p38 MAPK inhibitors SB 202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole) and SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H- imidaz ole) blocked A2B receptor-mediated production of IL-8. These results indicate that extracellular adenosine can regulate ERK, c-Jun N-terminal kinase, and p38 MAPK signaling cascades and that activation of ERK and p38 MAPK pathways are essential steps in adenosine A2B receptor-dependent stimulation of IL-8 production in HMC-1.