We have identified a mammalian protein called GIPC (for GAIP interacting protein, C terminus), which has a central PDZ domain and a C-terminal acyl carrier protein (ACP) domain. The PDZ domain of GIPC specifically interacts with RGS-GAIP, a GTPase-activating protein (GAP) for G␣ i subunits recently localized on clathrin-coated vesicles. Analysis of deletion mutants indicated that the PDZ domain of GIPC specifically interacts with the C terminus of GAIP (11 amino acids) in the yeast two-hybrid system and glutathione S-transferase (GST)-GIPC pull-down assays, but GIPC does not interact with other members of the RGS (regulators of G protein signaling) family tested. This finding is in keeping with the fact that the C terminus of GAIP is unique and possesses a modified C-terminal PDZ-binding motif (SEA). By immunoblotting of membrane fractions prepared from HeLa cells, we found that there are two pools of GIPC-a soluble or cytosolic pool (70%) and a membrane-associated pool (30%). By immunof luorescence, endogenous and GFP-tagged GIPC show both a diffuse and punctate cytoplasmic distribution in HeLa cells ref lecting, respectively, the existence of soluble and membrane-associated pools. By immunoelectron microscopy the membrane pool of GIPC is associated with clusters of vesicles located near the plasma membrane. These data provide direct evidence that the C terminus of a RGS protein is involved in interactions specific for a given RGS protein and implicates GAIP in regulation of additional functions besides its GAP activity. The location of GIPC together with its binding to GAIP suggest that GAIP and GIPC may be components of a G protein-coupled signaling complex involved in the regulation of vesicular trafficking. The presence of an ACP domain suggests a putative function for GIPC in the acylation of vesicle-bound proteins.The recently discovered family of RGS proteins (for regulators of G protein signaling) act as GTPase-activating proteins (GAPs) that bind to ␣ subunits of heterotrimeric G proteins of the G␣ i ͞G␣ q subclass and enhance their GTPase activity (1-5), thus facilitating their deactivation. Moreover, some family members serve as effector antagonists that compete with effector for binding to G␣ subunits (4).Today more than 20 different mammalian proteins are known to contain the diagnostic RGS domain, far more than there are G␣ i ͞G␣ q subunits. Although their RGS domains are highly homologous (45-80%), the N and C termini of mammalian RGS proteins outside the common RGS domain share little sequence similarity, suggesting that they have specific interactions and functions.Previously we have identified and characterized RGS-GAIP (6, 7), a RGS family member shown to directly interact with G␣ i3 through its RGS domain (6). We subsequently showed that GAIP is membrane-anchored (7) and, quite recently, that it is located on clathrin-coated vesicles and not on the plasma membrane (8). The localization of GAIP to clathrin-coated vesicles suggests GAIP may participate in the regulation of G protein-me...

GIPC is a PDZ protein located on peripheral endosomes that binds to the juxtamembrane region of the TrkA nerve growth factor (NGF) receptor and has been implicated in NGF signaling. We establish here that endogenous GIPC binds to the C terminus of APPL, a Rab5 binding protein, which is a marker for signaling endosomes. When PC12(615) cells are treated with either NGF or antibody agonists to activate TrkA, GIPC and APPL translocate from the cytoplasm and bind to incoming, endocytic vesicles carrying TrkA concentrated at the tips of the cell processes. GIPC, but not APPL, dissociates from these peripheral endosomes prior to or during their trafficking from the cell periphery to the juxtanuclear region, where they acquire EEA1. GIPC's interaction with APPL is essential for recruitment of GIPC to peripheral endosomes and for TrkA signaling, because a GIPC PDZ domain mutant that cannot bind APPL or APPL knockdown with small interfering RNA inhibits NGF-induced GIPC recruitment, mitogen-activated protein kinase activation, and neurite outgrowth. GIPC is also required for efficient endocytosis and trafficking of TrkA because depletion of GIPC slows down endocytosis and trafficking of TrkA and APPL to the early EEA1 endosomes in the juxtanuclear region. We conclude that GIPC, following its recruitment to TrkA by APPL, plays a key role in TrkA trafficking and signaling from endosomes.

NGF initiates the majority of its neurotrophic effects by promoting the activation of the tyrosine kinase receptor TrkA. Here we describe a novel interaction between TrkA and GIPC, a PDZ domain protein. GIPC binds to the juxtamembrane region of TrkA through its PDZ domain. The PDZ domain of GIPC also interacts with GAIP, an RGS (regulators of G protein signaling) protein. GIPC and GAIP are components of a G protein-coupled signaling complex thought to be involved in vesicular trafficking. In transfected HEK 293T cells GIPC, GAIP, and TrkA form a coprecipitable protein complex. Both TrkA and GAIP bind to the PDZ domain of GIPC, but their binding sites within the PDZ domain are different. The association of endogenous GIPC with the TrkA receptor was confirmed by coimmunoprecipitation in PC12 (615) cells stably expressing TrkA. By immunofluorescence GIPC colocalizes with phosphorylated TrkA receptors in retrograde transport vesicles located in the neurites and cell bodies of differentiated PC12 (615) cells. These results suggest that GIPC, like other PDZ domain proteins, serves to cluster transmembrane receptors with signaling molecules. When GIPC is overexpressed in PC12 (615) cells, NGF-induced phosphorylation of mitogen-activated protein (MAP) kinase (Erk1/2) decreases; however, there is no effect on phosphorylation of Akt, phospholipase C-gamma1, or Shc. The association of TrkA receptors with GIPC and GAIP plus the inhibition of MAP kinase by GIPC suggests that GIPC may provide a link between TrkA and G protein signaling pathways.

K3E/R14E is a dominant negative mutant of PLB that disrupts the structural integrity and function of the endogenous PLB and consequently enhances SERCA2 activity and myocyte contractility. In neonatal myocytes, the decrease in steady-state abundance of PLB by asPLB also leads to increased SERCA2 activity.

Ophthalmic drugs are delivered to ocular tissues predominantly via relatively simple formulations, such as topically dosed water-soluble drug solutions and water-insoluble drug suspensions in ointments. An ideal topical drug delivery system should possess certain desirable properties, such as good corneal and conjunctival penetration, prolonged precorneal residence time, easy instillation, non-irritative and comfortable to minimize lachrymation and reflex blinking, and appropriate rheological properties. In general, ocular efficacy is closely related to ocular drug bioavailability, which may be enhanced by increasing corneal drug penetration and prolonging precorneal drug residence time. To improve ocular bioavailability of topically dosed ophthalmic drugs, a variety of ocular drug delivery systems, such as hydrogels, microparticles, nanoparticles, microemulsions, liposomes and collagen shields, have been designed and investigated. These newer systems may, to some extent, control drug release and maintain therapeutic levels in ocular tissues over a prolonged period of time. This review focuses on the in vitro, ex vivo and in vivo studies of ophthalmic drugs formulated in nanoparticles published over the past two decades. The progress and development issues relating to ocular disposition, pharmacokinetics, efficacy and safety of the nanoparticle-formulated ophthalmic drugs are specifically addressed. Information and discussions summarized in this review are helpful for pharmaceutical scientists to develop better ophthalmic therapeutics.