Synaptojanin (synaptojanin 1) is a recently identified inositol 5-phosphatase, which is highly enriched in nerve terminals and is implicated in synaptic vesicle recycling. It is composed of three domains: an aminoterminal SacI homology region, a central inositol 5-phosphatase homology region, and a carboxyl-terminal proline-rich region. We have now identified and characterized a novel form of synaptojanin, synaptojanin 2, which has a broader tissue distribution. Synaptojanin 2 cDNA from rat brain library encodes a protein of 1,248 amino acids with a predicted M r of 138,268. The two synaptojanin isoforms share 57.2 and 53.8% amino acid identity in their SacI and phosphatase domains, respectively. In marked contrast, their carboxyl-terminal proline-rich regions bear little homology. Expression of synaptojanin 2 in COS7 cells produced a 140-kDa protein with inositol 5-phosphatase actvity. Protein binding assays demonstrated that among the major src homology 3-proteins known to bind to the proline-rich region of synaptojanin 1, Grb2, amphiphysin, and members of SH3p4/8/13 protein family, only Grb2 bound to that of synaptojanin 2. Furthermore, subcellular fractionation studies in transfected Chinese hamster ovary cells revealed that synaptojanin 2 was predominantly associated with the particulate fraction while synaptojanin 1 was mainly localized in the soluble fraction. This observation suggests that the proline-rich regions of synaptojanins 1 and 2 are implicated in different protein-protein interactions and direct the two isoforms to different subcellular compartments.Our results demonstrate the presence of a family of synaptojanin-type inositol 5-phosphatases with different tissue and subcellular distributions, which may be involved in distinct membrane trafficking and signal transduction pathways in mammalian cells.Inositol metabolism is now considered to play essential roles in membrane trafficking processes within the cell, apart from its well established role in signal transduction (1-3). Of particular importance, distinct phosphoinositides seem to be involved in specific vesicle transport steps, as demonstrated by critical requirement of several enzymes responsible for synthesis and metabolism of phosphoinositides in different membrane trafficking pathways (4).Synaptojanin is a recently identified inositol 5Ј-phosphatase enriched in nerve terminals (5-7). It has a 3-domain structure in which the central inositol 5Ј-phosphatase domain is flanked by a proline-rich domain at the carboxyl-terminal side and by a domain homologous to the yeast protein SacI (8) at the aminoterminal side. Synaptojanin is concentrated in nerve terminals and localized in close proximity to clathrin-and dynamincoated endocytic intermediates (9). Synaptojanin and dynamin 1 are the major interacting proteins for amphiphysin 1, another nerve terminal protein with a putative role in synaptic vesicle endocytosis (7, 10). In addition, synaptojanin, dynamin 1, and amphiphysin 1 undergo parallel dephosphorylation upon nerve terminal depolariz...

Several G-protein coupled receptors, such as the ␤1-adrenergic receptor (␤1-AR), contain polyproline motifs within their intracellular domains. Such motifs in other proteins are known to mediate protein-protein interactions such as with Src homology (SH)3 domains. Accordingly, we used the proline-rich third intracellular loop of the ␤1-AR either as a glutathione S-transferase fusion protein in biochemical ''pull-down'' assays or as bait in the yeast two-hybrid system to search for interacting proteins. Both approaches identified SH3p4͞p8͞p13 (also referred to as endophilin 1͞2͞3), a SH3 domain-containing protein family, as binding partners for the ␤1-AR. In vitro and in human embryonic kidney (HEK) 293 cells, SH3p4 specifically binds to the third intracellular loop of the ␤1-AR but not to that of the ␤2-AR. Moreover, this interaction is mediated by the C-terminal SH3 domain of SH3p4. Functionally, overexpression of SH3p4 promotes agonist-induced internalization and modestly decreases the Gs coupling efficacy of ␤1-ARs in HEK293 cells while having no effect on ␤2-ARs. Thus, our studies demonstrate a role of the SH3p4͞p8͞p13 protein family in ␤1-AR signaling and suggest that interaction between proline-rich motifs and SH3-containing proteins may represent a previously underappreciated aspect of G-protein coupled receptor signaling.

Interaction whereas the catalytic subunit (C) of both types of kinase appears essentially identical (1-3). The physiological relevance of cAMP-dependent protein phosphorylation and dephosphorylation in regulation of enzymes in key metabolic pathways has been clearly demonstrated (1, 4, 5), and additional advances have been made in understanding the effects of phosphorylation on many nonenzyme substrates (6, 7). The major recognized function of the regulatory subunit is inhibition of the kinase activity of the C present in the holoenzyme (1). The physiological significance of the existence of two types of cAMP-dependent protein kinase differing only in their regulatory subunit has not been fully elucidated. An increasing number of indications, however, suggest thatThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. the regulatory subunit may have other roles in addition to inhibition of C. (i) The concentration of regulatory subunits can be selectively increased several fold without any change in the concentration of C in certain cells in culture treated with dibutyryl cAMP (Bt2cAMP) or hormones that raise cAMP levels (reviewed in ref. 8). Under these conditions some cells appeared to acquire more differentiated characteristics, but the function of the excess regulatory subunit is not known. (ii) The protein kinase regulatory subunit seems capable of interacting with other cellular components in addition to C. The subcellular localization of both type I and type II cAMP-dependent protein kinases appears to be mediated by the attachment of the regulatory subunit to certain structural elements of the cell. A portion of both kinases have been recovered in particulate fractions of cell homogenates, and the ratio of the soluble/particulate kinase varied from one tissue to another (9-11). Kinase association with the particulate fraction was shown to be mediated by the regulatory subunit (either RI or RI,), since cAMP produced release of C but not the regulatory subunit into the soluble phase (10, 12). In addition, a specific association of RI, with microtubule-associated protein 2 (MAP2) (13,14) and with a calmodulin-binding multimeric complex (15) has been demonstrated. Finally, experiments using cDNA probes have allowed a direct demonstration of cAMP-mediated increases in specific mRNAs coding for a number of proteins (reviewed in ref. 8 SPreliminary results of this work were presented at the

The eisosome protein Pil1 interacts with the PI(4,5)P2 phosphatase Inp51, thereby recruiting it to the plasma membrane. Pil1 is essential for membrane localization of Inp51 but not for the homologous PI(4,5)P2 phosphatases Inp52 and Inp53. Consistent with this, Pil1 plays a crucial role in maintaining normal PI(4,5)P2 levels at the plasma membrane.