Natural populations of beach mice exhibit a characteristic color pattern, relative to their mainland conspecifics, driven by natural selection for crypsis. We identified a derived, charge-changing amino acid mutation in the melanocortin-1 receptor (Mc1r) in beach mice, which decreases receptor function. In genetic crosses, allelic variation at Mc1r explains 9.8% to 36.4% of the variation in seven pigmentation traits determining color pattern. The derived Mc1r allele is present in Florida's Gulf Coast beach mice but not in Atlantic coast mice with similar light coloration, suggesting that different molecular mechanisms are responsible for convergent phenotypic evolution. Here, we link a single mutation in the coding region of a pigmentation gene to adaptive quantitative variation in the wild.

Extracellular nucleotides (e.g., ATP) regulate many physiological and pathophysiological processes through activation of nucleotide (P2) receptors in the plasma membrane. Here we report that gene-targeted (knockout) mice that lack P2Y2 receptors have salt-resistant arterial hypertension in association with an inverse relationship between salt intake and heart rate, indicating intact baroreceptor function. Knockout mice have multiple alterations in their handling of salt and water: these include suppressed plasma renin and aldosterone concentrations, lower renal expression of the aldosterone-induced epithelial sodium channel alpha-ENaC, greater medullary expression of the Na-K-2Cl-cotransporter NKCC2, and greater furosemide-sensitive Na+ reabsorption in association with greater renal medullary expression of aquaporin-2 and vasopressin-dependent renal cAMP formation and water reabsorption despite similar vasopressin levels compared with wild type. Of note, smaller increases in plasma aldosterone were required to adapt renal Na+ excretion to restricted intake in knockout mice, suggesting a facilitation in renal Na+ retention. The results thus identify a previously unrecognized role for P2Y2 receptors in blood pressure regulation that is linked to an inhibitory influence on renal Na+ and water reabsorption. Based on these findings in knockout mice, we propose that a blunting in P2Y2 receptor expression or activity is a new mechanism for salt-resistant arterial hypertension.

Multistep proteolytic mechanisms are essential for converting proprotein precursors into active peptide neurotransmitters and hormones. Cysteine proteases have been implicated in the processing of proenkephalin and other neuropeptide precursors. Although the papain family of cysteine proteases has been considered the primary proteases of the lysosomal degradation pathway, more recent studies indicate that functions of these enzymes are linked to specific biological processes. However, few protein substrates have been described for members of this family. We show here that secretory vesicle cathepsin L is the responsible cysteine protease of chromaffin granules for converting proenkephalin to the active enkephalin peptide neurotransmitter. The cysteine protease activity was identified as cathepsin L by affinity labeling with an activity-based probe for cysteine proteases followed by mass spectrometry for peptide sequencing. Production of T he biosynthesis of enkephalin opioid peptides as well as numerous peptide neurotransmitters and hormones requires proteolytic processing of respective proprotein precursors within regulated secretory vesicles (1-4). The mature, processed enkephalin peptide is stored within these vesicles and undergoes stimulated secretion to mediate neurotransmission and cell-cell communication in the regulation of analgesia, behavior, and immune-cell functions. Secretory vesicles of neuroendocrine chromaffin cells (also known as chromaffin granules) contain enkephalin and its precursor proenkephalin (PE) (5, 6), with relevant prohormone convertases for converting PE into active enkephalin.The primary PE-cleaving activity in chromaffin granules has been characterized as a cysteine protease complex known as ''prohormone thiol protease'' (PTP) (7-10). The cysteine protease activity cleaves PE and enkephalin-containing peptide substrates at paired basic residues, as well as at certain monobasic residues, to generate appropriate enkephalin-related peptide products. Cellular inhibition of PTP by a cysteine protease inhibitor results in reduced production of enkephalin (11). Molecular identification of the protease component responsible for this cysteine protease activity will facilitate our understanding of multiple proteolytic enzymes that produce active peptides including the opioid [Met]enkephalin (ME) (12,13).In this study the protease responsible for PE-cleaving activity in chromaffin granules was identified by using an activity-based probe for cysteine proteases (14, 15) combined with mass spectrometry (MS) for peptide sequencing. Results identified secretory vesicle cathepsin L as the enzyme responsible for the previously described PTP cysteine protease activity involved in enkephalin and neuropeptide production (7-10). Cathepsin L generated the active peptide ME by cleaving enkephalin-containing peptide substrates at native dibasic and monobasic sites. Notably, cathepsin L colocalized with ME in the regulated secretory pathway of chromaffin cells. In cathepsin L gene knockout (KO) mice (16-1...

A key factor in Alzheimer's disease (AD) is the beta-secretase activity that is required for the production of beta-amyloid (Abeta) peptide from its amyloid precursor protein (APP) precursor. In this study, the majority of Abeta secretion from neuronal chromaffin cells was found to occur via the regulated secretory pathway, compared with the constitutive secretory pathway; therefore, beta-secretase activity in the regulated secretory pathway was examined for the production and secretion of Abeta in chromaffin cells obtained from in vivo adrenal medullary tissue. The presence of Abeta(1-40) in APP-containing chromaffin vesicles, which represent regulated secretory vesicles, was demonstrated by radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography. These vesicles also contain Abeta(1-42), measured by RIA. Significantly, regulated secretion of Abeta(1-40) from chromaffin cells represented the majority of secreted Abeta (> 95% of total secreted Abeta), compared with low levels of constitutively secreted Abeta(1-40). These results indicate the importance of Abeta production and secretion in the regulated secretory pathway as a major source of extracellular Abeta. Beta-secretase activity in isolated chromaffin vesicles was detected with the substrate Z-Val-Lys-Met-/MCA (methylcoumarinamide) that contains the beta-secretase cleavage site. Optimum beta-secretase activity in these vesicles required reducing conditions and acidic pH (pH 5-6), consistent with the in vivo intravesicular environment. Evidence for cysteine protease activity was shown by E64c inhibition of Z-Val-Lys-Met-MCA-cleaving activity, and E64c inhibition of Abeta(1-40) production in isolated chromaffin vesicles. Chromatography resolved the beta-secretase activity into two distinct proteolytic pathways consisting of: (i) direct cleavage of the beta-secretase site at Met-/Asp by two cysteine proteolytic activities represented by peaks Il-A and Il-B, and (ii) an aminopeptidase-dependent pathway represented by peak I cysteine protease activity that cleaves between Lys-/Met, followed by Met-aminopeptidase that would generate the beta-secretase cleavage site. Treatment of chromaffin cells in primary culture with the cysteine protease inhibitor E64d reduced the production of the beta-secretase product, a 12-14 kDa C-terminal APP fragment. In addition, BACE 1 and BACE 2 were detected in chromaffin vesicles; BACE 1 represented a small fraction of total beta-secretase activity in these vesicles. These results illustrate that multiple cysteine proteases, in combination with BACE 1, contribute to beta-secretase activity in the regulated secretory pathway. These results complement earlier findings for BACE 1 as beta3-secretase for Abeta production in the constitutive secretory pathway that provides basal secretion of Abeta into conditioned media. These findings suggest that drug inhibition of several proteases may be required for reducing Abeta levels as a potential therapeutic approach for AD.

This study demonstrates that endopin 2 is a unique secretory vesicle serpin that displays cross-class inhibition of cysteine and serine proteases, indicated by effective inhibition of papain and elastase, respectively. Homology of the reactive site loop (RSL) domain of endopin 2, notably at P1-P1' residues, with other serpins that inhibit cysteine and serine proteases predicted that endopin 2 may inhibit similar proteases. Recombinant N-His-tagged endopin 2 inhibited papain and elastase with second-order rate constants (k(ass)) of 1.4 x 10(6) and 1.7 x 10(5) M(-1) s(-1), respectively. Endopin 2 formed SDS-stable complexes with papain and elastase, a characteristic property of serpins. Interactions of the RSL domain of endopin 2 with papain and elastase were indicated by cleavage of endopin 2 near the predicted P1-P1' residues by these proteases. Endopin 2 did not inhibit the cysteine protease cathepsin B, or the serine proteases chymotrypsin, trypsin, plasmin, and furin. Endopin 2 in neuroendocrine chromaffin cells was colocalized with the secretory vesicle component (Met)enkephalin by confocal immunonfluorescence microscopy, and was present in isolated secretory vesicles (chromaffin granules) from chromaffin cells as a glycoprotein of 72-73 kDa. Moreover, regulated secretion of endopin 2 from chromaffin cells was induced by nicotine and KCl depolarization. Overall, these results demonstrate that the serpin endopin 2 possesses dual specificity for inhibiting both papain-like cysteine and elastase-like serine proteases. These findings demonstrate that endopin 2 inhibitory functions may occur in the regulated secretory pathway.

Several cell types, including cardiac myocytes and vascular endothelial cells, produce nitric oxide (NO) via both constitutive and inducible isoforms of NO synthase. NO attenuates cardiac contractility and contributes to contractile dysfunction in heart failure, although the precise molecular mechanisms for these effects are poorly defined. Adenylyl cyclase (AC) isoforms type 5 and 6, which are preferentially expressed in cardiac myocytes, may be inhibited via a direct nitrosylation by NO. Because endothelial NO synthase (eNOS and NOS3), ␤-adrenergic (␤AR) receptors, and AC6 all can localize in lipid raft/caveolin-rich microdomains, we sought to understand the role of lipid rafts in organizing components of ␤AR-G s -AC signal transduction together with eNOS. Using neonatal rat cardiac myocytes, we found that disruption of lipid rafts with ␤-cyclodextrin inhibited forskolin-stimulated AC activity and cAMP production, eliminated caveolin-3-eNOS interaction, and increased NO production. ␤AR-and G s -mediated activation of AC activity were inhibited by ␤-cyclodextrin treatment, but prostanoid receptor-stimulated AC activity, which appears to occur outside caveolin-rich microdomains, was unaffected unless eNOS was overexpressed and lipid rafts were disrupted. An NO donor, SNAP, inhibited basal and forskolin-stimulated cAMP production in both native cardiac myocytes and cardiac myocytes and pulmonary artery endothelial cells engineered to overexpress AC6. These effects of SNAP were independent of guanylyl cyclase activity and were mimicked by overexpression of eNOS. The juxtaposition of eNOS with ␤AR and AC types 5 and 6 results in selective regulation of ␤AR by eNOS activity in lipid raft domains over other G s -coupled receptors localized in nonraft domains. Thus co-localization of multiple signaling components in lipid rafts provides key spatial regulation of AC activity.Seven membrane-spanning G protein-coupled receptors (GPCR) 1 signal via heterotrimeric G-proteins that regulate effector molecules that generate second messengers. Numerous GPCR, including ␤-adrenergic (␤AR) and certain prostanoid receptors, couple to G s to stimulate adenylyl cyclase (AC) activity and the generation of cyclic AMP (cAMP). In cardiac myocytes, increased cAMP levels change several aspects of cardiac function to enhance, for example, rate and force of contraction and the rate of relaxation. cAMP action primarily occurs via activation of protein kinase A, which alters intracellular Ca 2ϩ dynamics and contractile function by phosphorylating calcium channels, troponin I and phospholamban (1, 2). ␤AR, activated by catecholamines, is the predominant G s -coupled GPCR in cardiac myocytes, but other receptors are also capable of regulating AC activity in these cells (3). We and others have demonstrated that ␤ 1 AR are efficiently coupled to activation of AC in cardiac myocytes due to their high degree of co-localization in a membrane microdomain composed of lipid rafts or caveolae, where AC is preferentially expressed (3-6). ␤ 2 AR and prost...

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.

G-protein-coupled receptors (GPCRs) and post-GPCR signalling components are expressed at low overall abundance in plasma membranes, yet they evoke rapid, high-fidelity responses. Considerable evidence suggests that GPCR signalling components are organized together in membrane microdomains, in particular lipid rafts, enriched in cholesterol and sphingolipids, and caveolae, a subset of lipid rafts that also possess the protein caveolin, whose scaffolding domain may serve as an anchor for signalling components. Caveolae were originally identified based on their morphological appearance but their role in compartmentation of GPCR signalling has been primarily studied by biochemical techniques, such as subcellular fractionation and immunoprecipitation. Our recent studies obtained using both microscopic and biochemical methods with adult cardiac myocytes show expression of caveolin not only in surface sarcolemmal domains but also at, or close to, internal regions located at transverse tubules/sarcoplasmic reticulum. Other results show co-localization in lipid rafts/caveolae of AC (adenylyl cyclase), in particular AC6, certain GPCRs, G-proteins and eNOS (endothelial nitric oxide synthase; NOS3), which generates NO, a modulator of AC6. Existence of multiple caveolin-rich microdomains and their expression of multiple modulators of signalling strengthen the evidence that caveolins and lipid rafts/caveolae organize and regulate GPCR signal transduction in eukaryotic cells. Cellular organelles and plasma membrane microdomainsClassical descriptions of cellular anatomy define organelles and regions (e.g. nucleus, Golgi apparatus, mitochondria and plasma membrane) as discrete entities. Biochemical efforts to understand the intracellular functions of these entities have primarily involved the isolation and characterization of subcellular fractions. Substantial evidence, however, supports the view that such preparations are quite heterogeneous, with differences in protein and lipid composition as well as in expression of organelle-specific functional activities. This is particularly the case for anatomically distinct regions of the plasma membrane, which can be strikingly non-uniform in expression of molecular components and functional activities. For example, the apical and basolateral membranes of epithelial cells, luminal and abluminal membranes of endothelial cells and dendritic, axonal and cell body membranes of neurons all demonstrate differences in expression of protein and lipid components and in differentiated responses characteristic of those cell types.