Chemokines are chemotactic cytokines which play an important role in the migration of immune cells to distant tissues or compartments within tissues. These proteins have also been demonstrated to play a major role in cancer metastasis. The C-C chemokine receptor type 7 (CCR7) is a member of the chemokine receptor family. CCR7 along with its ligands CCL19 and CCL21 plays an important role in innate immune response by trafficking of lymphocytes. In cancer, tumour cells expressing CCR7 migrate to lymphoid organs and thus disseminate to other organs. Neutralizing the interactions between CCL21/CCR7 would therefore be expected to inhibit the progression and metastasis of many different types of cancer to regional lymph nodes or distant organs. Our objective was to identify a potent small molecule antagonist of CCR7 as a prelude to the investigation of the role of this axis in cancer metastasis. In this study, we provided a brief description of chemokines and their role in health and disease with an emphasis on the CCR7/CCL19/CCL21 axis, as well as identification of a CCR7 antagonist “hit”. The potency of the CCR7 antagonist “hit” was optimised by synthesizing different CCR7 antagonist analogues. The “hit” optimization process has led to discover the most active compound amongst a series of different analogues which have the ability to bind and block CCR7 receptor. The efficacy of the most active compound and other analogues were evaluated in vitro using a calcium flux assay which is based on detecting fluorescent light emitted upon release of calcium ions. To identify a suitable cell line, which expresses CCR7 and capably respond to it, amongst a panel of cell lines for in vitro assessment of potency of synthesised compounds, we used Western blot assay and later by flow cytometry assay. The activity and selectivity of the most effective compound against CCR7 receptor was evaluated in vitro by other functional assays such as “configured agarose spot assay” and scratch assay. We first configured the existing under agarose assay to fulfil our requirements and then used it to assess activity and selectivity of compounds. The configured agarose spot assay also describes the application of the agarose spot for evaluation of cells chemotactic response to multiple chemokines under identical experiment conditions.

<p>The alpha-2 adrenergic receptor (α₂-AR) subfamily includes three different subtypes, α_2A-, α_2B- and α_2C-AR, all believed to exert their function through heterotrimeric G_i/o-proteins. The present study was undertaken in order to investigate subtype differences in terms of cellular response and to explore other potential signalling pathways of α₂-ARs.</p><p>Evidence is provided for a strong G_s-protein coupling capability of the α_2B-AR, leading to stimulation of adenylyl cyclase (AC). The difference between the α_2A- and α_2B-AR subtypes, in this respect, was shown to be due to differences in the second intracellular loops of the receptor proteins. Substitution of the second loop in α_2A-AR with the corresponding domain of α_2B-AR enrolled the chimeric α_2A/α_2B receptor with functional α_2B-AR properties. Dual G_i and G_s coupling can have different consequences for AC output. Using coexpression of receptors and G-proteins, it was shown that the ultimate cellular response of α_2B-AR activation is largely dependent on the ratio of G_i- to G_s-protein amounts in the cell. Also G_i- and G_o-proteins appear to have different regulatory influences on AC. Heterologous expression of AC2 together with G_i or G_o and the α_2A-AR resulted in receptor-mediated inhibition of protein kinase C-stimulated AC2 activity through G_o, whereas activation of G_i potentiated the activity. </p><p>α₂-ARs mobilize Ca²⁺ in response to agonists in some cell types. This response was shown to depend on tonic purinergic receptor activity in transfected CHO cells. Elimination of the tonic receptor activity almost completely inhibited the Ca²⁺ response of α₂-ARs.</p><p>In conclusion, α₂-ARs can couple to multiple G-proteins, including G_i, G_o and G_s. The cellular response to α₂-AR activation depends on which receptor subtype is expressed, which cellular signalling constituents are engaged (G-proteins and effectors), and the signalling status of the effectors (dormant or primed).</p>

<p>Communication between neuronal and non-neuronal is called volume transmission when the released neurotransmitter (NT) acts via diffusion and affects several target cells. Both the neurosecretory and postsynaptic cell responses are linked to [Ca²⁺]_i elevations. </p><p>In the present thesis the role of pre-and postsynaptic Ca²⁺ elevations has been investigated in the reconstituted "synapse" model comprised of NGF-differentiated PC12 and HEL cells as well as in SH-SY5Y neuroblastoma cells. In PC12 cells, both 70mM K⁺ and nicotine triggered NT release, which could be detected as a secondary [Ca²⁺]_i increase in surrounding HEL cells. Both secretagogues shared the same voltage-dependent Ca²⁺ influx pathway as judged from the pharmacological profile blockers of voltage-gated Ca²⁺ channels. The coupling of electrical responses to the activation of Ca²⁺ signaling via muscarinic receptors in SH-SY5Y cells was also studied. These data revealed that depolarization caused a considerable potentiation of the muscarinic Ca²⁺ response. The potentiated Ca²⁺ increase was mainly dependent on the enhanced Ca²⁺ influx and to a lesser extent on [Ca²⁺]_i release from intracellular stores. A phospholipase C (PLC) activator, m-3M3FBS was used to further study the role of G-protein coupled receptor (GPCR)-coupled Ca²⁺ signaling. However, it was found that m-3M3FBS instead triggered [Ca²⁺]_i elevations independently of PLC activation. </p><p>In conclusion, the results indicate that the magnitude of NT release from PC12 cells is sufficient to cause a robust activation of neighboring target cells. Postsynaptic muscarinic signaling is amplified due to integration of electrical excitation and GPCR signaling. The PLC activator, m-3M3FBS is not suitable for studies of PLC-mediated signals in intact cells.</p>

<p>Neuropeptide Y (NPY) is an abundant neurotransmitter in the nervous system and forms a family of evolutionarily related peptides together with peptide YY (PYY), pancreatic polypeptide (PP) and polypeptide Y (PY). These peptides are ligands to a family of receptors that mediate a wide range of physiological effects including stimulation of appetite. This work describes the molecular cloning of four novel NPY receptors.</p><p>In rat a receptor called PP1, later renamed Y₄, was cloned and characterized. It displays the highest amino acid sequence identity to the Y₁ receptor. Rat Y₄ differs extensively from human Y₄, cloned subsequently, in both pharmacological properties, tissue distribution, and amino acid sequence with only 75% identity. Rat and human Y₄are the most diverged orthologues in the NPY receptor family.</p><p>In guinea pig, the y₆ receptor gene was found to be a pseudogene with several frameshift mutations. The gene is a pseudogene in human and pig too, but seems to give rise to a functional receptor in mouse and rabbit. This unusual evolutionary situa- tion may be due to inactivation of the gene in a mammalian ancestor and then restoration of expression in mouse and rabbit, but perhaps more likely due to independent inactivations in guinea pig, human and pig.</p><p>In zebrafish, two new intronless receptor genes were cloned. Sequence comparisons suggest that both receptors are distinct from the mammalian receptors Y₁, Y₄ and y₆, hence they were named Ya and Yb. Chromosomal localization provides further support that Ya and Yb may be distinct subtypes. </p><p>The discoveries of the rat Y₄ and zebrafish Ya and Yb receptors were unexpected and show that the NPY receptor family is larger than previously thought.</p>

The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.

Neuropeptide Y (NPY) is an abundant neurotransmitter in the nervous system and forms a family of evolutionarily related peptides together with peptide YY (PYY), pancreatic polypeptide (PP) and polypeptide Y (PY). These peptides are ligands to a family of receptors that mediate a wide range of physiological effects including stimulation of appetite. This work describes the molecular cloning of four novel NPY receptors. In rat a receptor called PP1, later renamed Y4, was cloned and characterized. It displays the highest amino acid sequence identity to the Y1 receptor. Rat Y4 differs extensively from human Y4, cloned subsequently, in both pharmacological properties, tissue distribution, and amino acid sequence with only 75% identity. Rat and human Y4 are the most diverged orthologues in the NPY receptor family. In guinea pig, the y6 receptor gene was found to be a pseudogene with several frameshift mutations. The gene is a pseudogene in human and pig too, but seems to give rise to a functional receptor in mouse and rabbit. This unusual evolutionary situa- tion may be due to inactivation of the gene in a mammalian ancestor and then restoration of expression in mouse and rabbit, but perhaps more likely due to independent inactivations in guinea pig, human and pig. In zebrafish, two new intronless receptor genes were cloned. Sequence comparisons suggest that both receptors are distinct from the mammalian receptors Y1, Y4 and y6, hence they were named Ya and Yb. Chromosomal localization provides further support that Ya and Yb may be distinct subtypes. The discoveries of the rat Y4 and zebrafish Ya and Yb receptors were unexpected and show that the NPY receptor family is larger than previously thought.

The alpha-2 adrenergic receptor (α2-AR) subfamily includes three different subtypes, α2A-, α2B- and α2C-AR, all believed to exert their function through heterotrimeric Gi/o-proteins. The present study was undertaken in order to investigate subtype differences in terms of cellular response and to explore other potential signalling pathways of α2-ARs. Evidence is provided for a strong Gs-protein coupling capability of the α2B-AR, leading to stimulation of adenylyl cyclase (AC). The difference between the α2A- and α2B-AR subtypes, in this respect, was shown to be due to differences in the second intracellular loops of the receptor proteins. Substitution of the second loop in α2A-AR with the corresponding domain of α2B-AR enrolled the chimeric α2A/α2B receptor with functional α2B-AR properties. Dual Gi and Gs coupling can have different consequences for AC output. Using coexpression of receptors and G-proteins, it was shown that the ultimate cellular response of α2B-AR activation is largely dependent on the ratio of Gi- to Gs-protein amounts in the cell. Also Gi- and Go-proteins appear to have different regulatory influences on AC. Heterologous expression of AC2 together with Gi or Go and the α2A-AR resulted in receptor-mediated inhibition of protein kinase C-stimulated AC2 activity through Go, whereas activation of Gi potentiated the activity. α2-ARs mobilize Ca2+ in response to agonists in some cell types. This response was shown to depend on tonic purinergic receptor activity in transfected CHO cells. Elimination of the tonic receptor activity almost completely inhibited the Ca2+ response of α2-ARs. In conclusion, α2-ARs can couple to multiple G-proteins, including Gi, Go and Gs. The cellular response to α2-AR activation depends on which receptor subtype is expressed, which cellular signalling constituents are engaged (G-proteins and effectors), and the signalling status of the effectors (dormant or primed).

Communication between neuronal and non-neuronal is called volume transmission when the released neurotransmitter (NT) acts via diffusion and affects several target cells. Both the neurosecretory and postsynaptic cell responses are linked to [Ca2+]i elevations. In the present thesis the role of pre-and postsynaptic Ca2+ elevations has been investigated in the reconstituted "synapse" model comprised of NGF-differentiated PC12 and HEL cells as well as in SH-SY5Y neuroblastoma cells. In PC12 cells, both 70mM K+ and nicotine triggered NT release, which could be detected as a secondary [Ca2+]i increase in surrounding HEL cells. Both secretagogues shared the same voltage-dependent Ca2+ influx pathway as judged from the pharmacological profile blockers of voltage-gated Ca2+ channels. The coupling of electrical responses to the activation of Ca2+ signaling via muscarinic receptors in SH-SY5Y cells was also studied. These data revealed that depolarization caused a considerable potentiation of the muscarinic Ca2+ response. The potentiated Ca2+ increase was mainly dependent on the enhanced Ca2+ influx and to a lesser extent on [Ca2+]i release from intracellular stores. A phospholipase C (PLC) activator, m-3M3FBS was used to further study the role of G-protein coupled receptor (GPCR)-coupled Ca2+ signaling. However, it was found that m-3M3FBS instead triggered [Ca2+]i elevations independently of PLC activation. In conclusion, the results indicate that the magnitude of NT release from PC12 cells is sufficient to cause a robust activation of neighboring target cells. Postsynaptic muscarinic signaling is amplified due to integration of electrical excitation and GPCR signaling. The PLC activator, m-3M3FBS is not suitable for studies of PLC-mediated signals in intact cells.

G protein-coupled receptors (GPCRs) are one of the largest protein families in mammals. GPCRs are instrumental for hormonal and neurotransmitter signalling and are important in all major physiological systems of the body. Paper I describes the repertoire of GPCRs in Branchiostoma floridae, which is one of the species most closely related species to vertebrates. Mining and phylogenetic analysis of the amphioxus genome showed the presence of at least 664 distinct GPCRs distributed among all the main families of GPCRs; Glutamate (18), Rhodopsin (570), Adhesion (37), Frizzled (6) and Secretin (16). Paper II contains studies of the Adhesion, Methuselah and Secretin GPCR families in nine genomes. The Adhesion GPCRs are the most complex gene family among GPCRs with large genomic size, multiple introns and a fascinating flora of functional domains. Phylogenetic analysis showed Adhesion group V (that contains GPR133 and GPR144) to be the closest relative to the Secretin family among the groups in the Adhesion family, which was also supported by splice site setup and conserved motifs. Paper III examines the repertoire of human transmembrane proteins. These form key nodes in mediating the cell’s interaction with the surroundings, which is one of the main reasons why the majority of drug targets are membrane proteins. We identified 6,718 human membrane proteins and classified the majority of them into 234 families of which 151 belong to the three major functional groups; Receptors (63 groups, 1,352 members), Transporters (89 groups, 817 members) or Enzymes (7 groups, 533 members). In addition, 74 Miscellaneous groups were shown to include 697 members. Paper IV clarifies the hierarchy of the main families and evolutionary origin of majority of the metazoan GPCR families. Overall, it suggests common decent of at least 97% of the GPCRs sequences found in humans, including all the main families.

Questions have been raised about the genomic complexity of the human genome, since it was reported that it only consisted of 32,000 genes. Alternative splicing is considered the explanation of the enormous difference between the number of genes and the number of proteins. Aligning expressed sequence tags (ESTs) to the genomic sequence has become a popular approach for gene prediction, revealing alternative splice variants. The aim in this thesis is to identify and analyse splice variants of the adhesion family of G protein-coupled receptors using EST data. 75% of the genes in the data set of 33 sequences were found to have a total of 51 splice variants. About half of the variants were considered functional.