<p>Neuropeptide Y (NPY) is known to influence a vast number of physiological and behavioral processes such as vasoconstriction, circadian rhythms, feeding, anxiety and memory. Peptides of the NPY family bind to five different cloned G-protein coupled receptor subtypes (Y1, 2, 4-6). The studies compiled in this thesis present inter-species comparisons of sequence similarities, binding properties and expression patterns among receptors of the NPY family.</p><p>Cloning of Y1 and Y2 receptor subtypes from guinea pigs revealed strong binding profile similarity to the corresponding human receptors. Previously demonstrated atypical binding profiles in the caval vein of guinea pigs were concluded to result from other receptors than the cloned Y1 and Y2 receptors, or possibly combinations of distinct receptor subtypes.</p><p>The guinea pig Y5 receptor was found to be expressed in regions of the brain that have been indicated as important for regulation of food intake. Expression in the hypothalamus, amygdala and brain stem was noticed, similar to studies in rats and humans. In other brain regions, such as the striatum and hippocampus, some species differences were observed.</p><p>Mutagenesis studies of the human Y1 receptor indicated sites important for binding both of endogenous agonists and synthetic antagonists. Putative new sites of interaction with the Y1 antagonists BIBP3226 and/or SR120819A were recognized. The data were used to construct a three-dimensional structure model, based on a high-resolution bovine rhodopsin model.</p><p>Cloning of the chicken (<i>Gallus gallus</i>) Y1, Y2 and Y5 receptors revealed high sequence similarities with mammalian receptors. Most endogenous ligands bound with similar affinities as to mammalian receptors. The strongest exception was the discovery of high-affinity binding to chicken Y2 of [Leu³¹, Pro³⁴]NPY, which was previously considered to bind non-Y2 receptors only. </p><p>The new human Y1 receptor model provides a basis for further investigations of ligand-receptor interactions which will be aided by information on NPY receptors from other taxa. Guinea pigs are concluded to be a good complement to rats and mice for studying NPY signaling. These results demonstrate the benefits of species comparisons for pharmacological studies.</p>

Neuropeptide Y (NPY) is known to influence a vast number of physiological and behavioral processes such as vasoconstriction, circadian rhythms, feeding, anxiety and memory. Peptides of the NPY family bind to five different cloned G-protein coupled receptor subtypes (Y1, 2, 4-6). The studies compiled in this thesis present inter-species comparisons of sequence similarities, binding properties and expression patterns among receptors of the NPY family. Cloning of Y1 and Y2 receptor subtypes from guinea pigs revealed strong binding profile similarity to the corresponding human receptors. Previously demonstrated atypical binding profiles in the caval vein of guinea pigs were concluded to result from other receptors than the cloned Y1 and Y2 receptors, or possibly combinations of distinct receptor subtypes. The guinea pig Y5 receptor was found to be expressed in regions of the brain that have been indicated as important for regulation of food intake. Expression in the hypothalamus, amygdala and brain stem was noticed, similar to studies in rats and humans. In other brain regions, such as the striatum and hippocampus, some species differences were observed. Mutagenesis studies of the human Y1 receptor indicated sites important for binding both of endogenous agonists and synthetic antagonists. Putative new sites of interaction with the Y1 antagonists BIBP3226 and/or SR120819A were recognized. The data were used to construct a three-dimensional structure model, based on a high-resolution bovine rhodopsin model. Cloning of the chicken (Gallus gallus) Y1, Y2 and Y5 receptors revealed high sequence similarities with mammalian receptors. Most endogenous ligands bound with similar affinities as to mammalian receptors. The strongest exception was the discovery of high-affinity binding to chicken Y2 of [Leu31, Pro34]NPY, which was previously considered to bind non-Y2 receptors only. The new human Y1 receptor model provides a basis for further investigations of ligand-receptor interactions which will be aided by information on NPY receptors from other taxa. Guinea pigs are concluded to be a good complement to rats and mice for studying NPY signaling. These results demonstrate the benefits of species comparisons for pharmacological studies.

This work identified an ortholog of Abp1 (actin binding protein 1) in Dictyostelium (Dabp1). In order to analyze the functions of Dabp1 in Dictyostelium, loss-of–function studies and gain-of-function studies were performed by generating cells that either deleted the Dabp1 gene from the genome or overexpressed the Dabp1 protein. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild type cells had a single leading pseudopodium and a single uropod, whereas more than 27% of Dabp1 null cells projected multiple pseudopodia. Similarly, ~ 90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium. This work also revealed an interplay between Dabp1 and MyoB, one of the Myosin I proteins, in controlling pseudopodia formation in Dictyostelium. These two proteins colocalize partially at the cortex in growing cells. The peripheral localization of MyoB was dependent on Dabp1. Depletion of both Dabp1 and MyoB caused defects in organization of the actin cytoskeleton and actin related activities such as formation of small F-actin filled spikes on the cell cortex of growing cells, a higher percentage of multinucleated cells, and an increased number of pseudopodia branching extensively. When MyoB was overexpressed in Dabp1 null mutants, cells had similar phenotypes as Dabp1/MyoB double null mutants, and displayed an increased number of pseudopodia with many branches. Overexpression of Dabp1 in MyoB null mutants rescued the defects in pseudopodia formation. The SH3 of Dabp1 was shown to be important for the rescue of defects caused by depletion of MyoB. Collectively, these data suggest that MyoB and Dabp1 work cooperatively to regulate the uniformity and integrity of the actin extensions during chemotaxis. MyoB requires Dabp1 to function in this process. Dabp1 may function as a scaffold to recruit MyoB to the proper localization. These studies of Dabp1 in Dictyostelium raise broad question about functions of actinassociated proteins in pseudopodia formation and the importance of uniformity and integrity for actin structures in chemotaxis. / text