We previously demonstrated that mouse sperm capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca2+, and NaHCO(3), all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO-(4) to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO-(4) and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO-(3) is necessary for the capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO-(3) or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO-(3) effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm.

Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk. The activation of this signaling pathway, as well as capacitation, requires bovine serum albumin (BSA) in the incubation medium. BSA is hypothesized to modulate capacitation through its ability to remove cholesterol from the sperm plasma membrane. Here we demonstrate that the cholesterol-binding heptasaccharides, methyl-␤-cyclodextrin and OH-propyl-␤-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these ␤-cyclodextrins were also demonstrated to increase protein tyrosine phosphorylation in the absence of BSA in both mouse and bull sperm, and the patterns of phosphorylation were similar to those induced by media containing BSA. The potency of the different ␤-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the ␤-cyclodextrins with cholesterol-SO 4 Ϫ to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The ␤-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO 3 and protein kinase A-dependent. The ␤-cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the zona pellucida to induce the acrosome reaction and by successful fertilization in vitro. In summary, ␤-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further support the coupling of cholesterol efflux to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems, thus defining a new mode of cellular signal transduction initiated by cholesterol release.

Degeneration of neurons in Alzheimer's disease is mediated by ␤-amyloid peptide by diverse mechanisms, which include a putative apoptotic component stimulated by unidentified signaling events. This report describes a novel ␤-amyloid peptide-binding protein (denoted BBP) containing a G protein-coupling module. BBP is one member of a family of three proteins containing this conserved structure. The BBP subtype bound human ␤-amyloid peptide in vitro with high affinity and specificity. Expression of BBP in cell culture induced caspase-dependent vulnerability to ␤-amyloid peptide toxicity. Expression of a signaling-deficient dominant negative BBP mutant suppressed sensitivity of human Ntera-2 neurons to ␤-amyloid peptide mediated toxicity. These findings suggest that BBP is a target of neurotoxic ␤-amyloid peptide and provide new insight into the molecular pathophysiology of Alzheimer's disease.Genetic and biochemical data have coalesced to establish that ␤-amyloid peptide (A␤) 1 is a causative factor in neuron death and the consequent dimunition of cognitive abilities observed in Alzheimer's disease (1, 2). Plasma lipoproteins and their cell surface receptors influence sequestration and clearance of soluble A␤, contributing to the etiology of the disease (3-6). Inflammatory responses and oxidative damage also appear to contribute to the loss of neurons in Alzheimer's disease (7-10). Although the earliest cellular perturbations remain unclear, recent findings indicate that A␤ may act as an initiating factor in the death of neurons by inducing signaling pathways leading to apoptosis (11-17). However, the specific molecular target(s) transducing these A␤ effects has not been identified. The intracellular protein ERAB can bind A␤ in vitro, and neuroblastoma cells expressing recombinant ERAB undergo apoptosis when treated with exogenously added A␤ (18), but the mechanism by which ERAB may affect apoptotic signaling remains obscure. We identified a novel human ␤-amyloid peptide binding protein (BBP) utilizing yeast 2-hybrid technology. Analysis of the BBP amino acid sequence revealed the presence of a structural module related to that of the 7 transmembrane domain G protein-coupled receptor superfamily and known to be important in heterotrimeric G protein activation. Data suggest that BBP mediates cellular vulnerability to A␤ toxicity through a G protein-regulated program of cell death. Two related proteins (BLP1, BLP2; BBP-like proteins) were identified by sequence and structural similarities to BBP, but only the BBP subtype regulates a response to A␤. EXPERIMENTAL PROCEDURESYeast Two-hybrid Systems-Yeast 2-hybrid (Y2H) expression plasmids were constructed in the vectors pAS2 and pACT2 (19). Strain CY770 (20) served as host for Y2H assays. Sequences encoding A␤ 42 were amplified by PCR using primers incorporating restriction sites for subsequent ligation into pAS2, using a human APP (amyloid precursor protein) cDNA clone as template. A Y2H plasmid library consisting of cDNA fragments isolated from human fetal brain clone...

SR 121463A, a potent and selective, orally active, nonpep-tide vasopressin V 2 receptor antagonist, has been characterized in several in vitro and in vivo models. This compound displayed highly competitive and selective affinity for V 2 receptors in rat, bovine and human kidney (0.6 K i [nM] 4.1). In this latter preparation, SR 121463A potently antagonized arginine vasopressin (AVP)-stimulated adenylyl cyclase activity (K i 0.26 0.04 nM) without any intrinsic agonistic effect. In autoradiographic experiments performed in rat kidney sections, SR 121463A displaced [ 3 H]AVP labeling especially in the medullo-papillary region and confirmed that it is a suitable tool for mapping V 2 receptors. In comparison, the nonpeptide V 2 antagonist, OPC-31260, showed much lower affinity for animal and human renal V 2 receptors and lower efficacy to inhibit vasopressin-stimulated adenylyl cyclase (K i in the 10 nanomolar range). Moreover , OPC-31260 exhibited a poor V 2 selectivity profile and can be considered as a V 2 /V 1a ligand. In normally hydrated conscious rats, SR 121463A induced powerful aquaresis after intravenous (0.003-0.3 mg/kg) or oral (0.03-10 mg/kg) administration. The effect was dose-dependent and lasted about 6 hours at the dose of 3 mg/kg p.o. OPC-31260 had a similar aquaretic profile but with markedly lower oral efficacy. The action of SR 121463A was purely aquaretic with no changes in urine Na and K excretions unlike that of known diuretic agents such as furosemide or hydrochloro-thiazide. In addition, no antidiuretic properties have been detected with SR 121463A in vasopressin-deficient Brattle-boro rats. Thus, SR 121463A is the most potent and selective , orally active V 2 antagonist yet described and could be a powerful tool for exploring V 2 receptors and the therapeutical usefulness of V 2 blocker aquaretic agents in water-retaining diseases. (J. Clin. Invest. 1996. 98:2729-2738.) Key words: SR 121463A • vasopressin • nonpeptide antagonist • V 2 receptor • aquaretic

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