Joseph K. Angleson

Dense Core Vesicle Dynamics in Caenorhabditis elegans Neurons and the Role of Kinesin UNC‐104

We have developed a model system in Caenorhabditis elegans to perform genetic and molecular analysis of peptidergic neurotransmission using green fluorescent protein (GFP)‐tagged IDA‐1. IDA‐1 represents the nematode ortholog of the transmembrane proteins ICA512 and phogrin that are localized to dense core secretory vesicles (DCVs) of mammalian neuroendocrine tissues. IDA‐1::GFP was expressed in a small subset of neurons and present in both axonal and dendritic extensions, where it was localized to small mobile vesicular elements that at the ultrastructural level corresponded to 50 nm electron‐dense objects in the neuronal processes. The post‐translational processing of IDA‐1::GFP in transgenic worms was dependent on the neuropeptide proprotein convertase EGL‐3, indicating that the protein was efficiently targeted to the peptidergic secretory pathway. Time‐lapse epifluorescence microscopy of IDA‐1::GFP revealed that DCVs moved in a saltatory and bidirectional manner. DCV velocity profiles exhibited multiple distinct peaks, suggesting the participation of multiple molecular motors with distinct properties. Differences between velocity profiles for axonal and dendritic processes furthermore suggested a polarized distribution of the molecular transport machinery. Study of a number of candidate mutants identified the kinesin UNC‐104 (KIF1A) as the microtubule motor that is specifically responsible for anterograde axonal transport of DCVs at velocities of 1.6 μm/s−2.7 μm/s.

Retention and stimulus-dependent recycling of dense core vesicle content in neuroendocrine cells

We have used fluorescence imaging of individual exocytic events in combination with immunogold electron microscopy and FM1-43 photoconversion to study the stimulus-dependent recycling of dense core vesicle content in isolated rat pituitary lactotrophs. Secretory stimulation with high external [K+] resulted in 100 exocytic sites per cell that were labeled by extracellular antibodies against the peptide hormone prolactin. Morphological analysis demonstrated that the prolactin was retained and internalized in intact dense cores. Vesicles containing non-secreted, internalized prolactin did not colocalize with DiI-LDL that had been chased into lysosomes but did transiently colocalize with internalized transferrin. The recycling vesicles also trafficked through a syntaxin 6-positive compartment but not the TGN38-positive trans-Golgi. Recycling vesicles, which returned to the cell surface in a slow basal manner, could also be stimulated to undergo exocytosis with a high release probability during subsequent exocytic stimulation with external K+. These studies suggest a functional role for recycling vesicles that retain prolactin.

Functional expression of frog and rainbow trout melanocortin 2 receptors using heterologous MRAP1s

Analysis of the functional expression of the melanocortin 2 receptor (MC2R) from a rather broad spectrum of vertebrates indicates that MC2R is exclusively selective for the ligand, ACTH, and the melanocortin receptor accessory protein 1 (MRAP1) is required for high affinity ACTH binding and activation of MC2R. A phylogenetic analysis of MRAP1 suggested that tetrapod sequences and bony fish sequences may represent two distinct trends in the evolution of the mrap1 gene. To test this hypothesis, a frog (Xenopus tropicalis) MC2R was expressed in CHO cells either in the presence of a tetrapod (mouse) MRAP1 or a bony fish (zebrafish) MRAP1. The response of frog MC2R to different concentrations of human ACTH(1-24) was more robust in the presence of mouse MRAP1 than in the presence of zebrafish MRAP1. Conversely, the cAMP response mediated by the rainbow trout (Oncorhynchus mykiss) MC2R was almost twofold higher and occurred at 1000-fold lower ACTH concentration in the presence of zebrafish MRAP1 than in the presence of mouse MRAP1. Collectively, these experiments raise the possibility that at least two distinct trends have emerged in the co-evolution of MC2R/MRAP1 interactions during the radiation of the vertebrates.

Sustained stimulation of exocytosis triggers continuous membrane retrieval in rat pituitary somatotrophs.

1 We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch‐clamp capacitance, FM1‐43 fluorescence imaging and amperometry. 2 Stimulation of exocytosis through voltage‐dependent Ca2+ channels by depolarizations (1‐5 s) increased the capacitance by 4.3 ± 0.9 % and the fluorescence by 6.6 ± 1.1 % (10 cells). The correlation between the capacitance and fluorescence changes indicated that the cell membrane and granule membrane added via exocytosis were stained with the membrane‐bound fluorescent dye FM1‐43 in a quantitatively similar manner. 3 Intracellular dialysis (0.5‐4.5 min) with elevated Ca2+ (1.5‐100 μm) evoked continuous exocytosis that was detected with a carbon fibre electrode from dopamine‐loaded cells (10 cells) or as an increase in FM1‐43 fluorescence (56 ± 10 %; 21 cells). Interestingly during Ca2+ dialysis the capacitance did not significantly change (2 ± 1 %; 31 cells), indicating that endocytosis efficiently retrieved increased cell membrane. 4 Sustained endocytosis was not blocked when the intracellular GTP (300 μm) was replaced with GTPγS. Replacing intracellular Ca2+ (100 μm) with Ba2+ (300 μm) or Sr2+ (200 μm), or reducing the pH of the intracellular solution from 7.2 to 6.2 did not block sustained endocytosis. 5 Our results suggest that pituitary somatotrophs have the ability to undergo continuous exocytosis and membrane retrieval that persist in whole‐cell recordings.

Modeling the evolution of the MC2R and MC5R genes: Studies on the cartilaginous fish, Heterondotus francisci

Comparative studies support the hypothesis that the proliferation of melanocortin receptor genes (MCRs) in gnathostomes corresponds to the 2R hypothesis for the radiation of gene families in Phylum Chordata. This mini-review will initially focus on the distribution of MCRs in cartilaginous fish and the relationship between the shark MC5R gene and the proposed ancestral MC5R/2R gene. This section will be followed by the results of recent studies on the features of the ligand binding site common to all melanocortin receptors. These data will provide the background for a set of hypotheses to explain the unique ligand selectivity of the MC2 receptor in teleosts and tetrapods.

Using the human melanocortin-2 receptor as a model for analyzing hormone/receptor interactions between a mammalian MC2 receptor and ACTH(1-24).

When considering the interactions between the melanocortin peptides (i.e., ACTH, α-MSH, β-MSH, γ-MSH) and the melanocortin receptors (i.e., MC1R, MC2R, MC3R, MC4R, MC5R), it appears that the structure/function relationship between ACTH and MC2R is the most complicated. Human ACTH(1-24) and the human melanocortin-2 receptor provide a useful model system for understanding how ACTH emerged as the sole ligand for the melanocortin-2 receptor of bony vertebrates. This review will discuss how studies utilizing analogs of hACTH(1-24) have revealed two critical amino acid motifs in this ligand (HFRW and KKRRP) which are required for activation of the melanocortin-2 receptor. In addition, observations on the unique activation features of the melanocortin-2 receptor, as revealed from studies on Familial Glucocorticoid Deficiency, will be considered. Finally, the evolutionary implications of the relationship between MC2R and MRAP1 will be discussed.

Evolution of melanocortin receptors in cartilaginous fish: Melanocortin receptors and the stress axis in elasmobranches

There is general agreement that the presence of five melanocortin receptor genes in tetrapods is the result of two genome duplications that occurred prior to the emergence of the gnathostomes, and at least one local gene duplication that occurred early in the radiation of the ancestral gnathostomes. Hence, it is assumed that representatives from the extant classes of gnathostomes (i.e., Chondrichthyes, Actinopterygii, Sarcopterygii) should also have five paralogous melanocortin genes. Current studies on cartilaginous fishes indicate that while there is evidence for five paralogous melanocortin receptor genes in this class, to date all five paralogs have not been detected in the genome of a single species. This mini-review will discuss the ligand selectivity properties of the melanocortin-3 receptor of the elephant shark (subclass Holocephali) and the ligand selectivity properties of the melanocortin-3 receptor, melanocortin-4 receptor, and the melanocortin-5 receptor of the dogfish (subclass Elasmobranchii). The potential relationship of these melanocortin receptors to the hypothalamus/pituitary/interrenal axis will be discussed.

Evolution of the melanocortin-2 receptor in tetrapods: studies on Xenopus tropicalis MC2R and Anolis carolinensis MC2R.

The tetrapods are a diverse assemblage of vertebrates, and that diversity is reflected in the sequences of tetrapod melanocortin-2 receptors (MC2Rs). In this review, the features common to human (mammal), Gallus gallus (bird), Anolis carolinensis (reptile), and Xenopus tropicalis (amphibian) MC2Rs in terms of ligand selectivity, requirements for interaction with MRAP1, and the effects of alanine substitutions to three amino acid motifs in the ligand hACTH(1-24) are discussed. Analysis of the effects of alanine substitutions to the H(6)F(7)R(8)W(9) and the K(15)K(16)R(17)R(18)P(19) motifs of hACTH(1-24) indicated that activation of A. carolinensis MC2R and X. tropicalis MC2R was more adversely affected by alanine substitutions at these positions as compared to the response of human MC2R to these same analogs. Furthermore, single alanine substitutions in the G(10)K(11)P(12)V(13)G(14) motif of hACTH(1-24) had negative affects on activation of both A. carolinensis MC2R and X. tropicalis MC2R that were not observed for human MC2R. The implications of responses to the various analogs of hACTH(1-24) in terms of the mechanism for mediating the activation of these various tetrapod melanocortin-2 receptors are discussed.

Observations on the ligand selectivity of the melanocortin 2 receptor.

The melanocortin 2 receptor (MC2R) is unique in terms of ligand selectivity and in vitro expression in mammalian cell lines as compared to the other four mammalian MCRs. It is well established that ACTH is the only melanocortin ligand that can activate the ACTH receptor (i.e., melanocortin 2 receptor). Recent studies have provided new insights into the presence of a common binding site for the HFRW motif common to all melanocortin ligands. However, the activation of the melanocortin 2 receptor requires an additional amino acid motif that is only found in the sequence of ACTH. This mini-review will focus on these two topics and provide a phylogenetic perspective on the evolution of MC2R ligand selectivity.

Recycling of intact dense core vesicles in neurites of NGF‐treated PC12 cells

Exocytic fusion in neuroendocrine cells does not always result in complete release of the peptide contents from dense core vesicles (DCVs). In this study, we use fluorescence imaging and immunoelectron microscopy to examine the retention, endocytosis and recycling of chromogranin B in DCVs of NGF‐treated PC12 cells. Our results indicate that DCVs retained and retrieved an intact core that was available for subsequent exocytic release. The endocytic process was inhibited by cyclosporine A or by substitution of extracellular Ca2+ with Ba2+ and the total recycling time was less than 5 min.