G protein-coupled receptors (GPCRs) control many physiological processes by converting input from the extracellular environment (e.g., light, neurotransmitters, hormones, or other ligands) into intracellular signal transduction. Due to their crucial role in various disease states and their tractability (e.g., predominant plasma membrane localization), GPCRs represent a major therapeutic class. Although 30-40% of the currently marketed drugs target GPCRs, only a small fraction of all human GPCRs have been targeted as of yet. CXCR4 is a member of the chemokine receptors, a family of GPCRs with a central role in the development and homeostasis of the immune system. Additionally, CXCR4 is overexpressed in many cancers, in which the receptor is implicated in oncogenic processes like proliferation and metastasis. In the last decades, it became clear that some GPCRs not only exist as monomeric entities but also form interactions among themselves or with other receptors, a process called oligomerization. These dimeric or higher-order oligomeric structures can be functionally distinct from monomeric ones. In artificial systems, CXCR4 switches from mainly monomeric at low expression levels to predominantly dimeric or higher-order oligomeric at high expression levels. The human cytomegalovirus (HCMV) is a widespread pathogen that has infected a large fraction of the population. In immunocompetent individuals, this herpesvirus establishes latent and asymptomatic infections. However, in organ-transplant or immunocompromised settings, HCMV may reactivate with severe consequences, sometimes even resulting in death. HCMV infections are associated with several malignancies, including glioblastoma. In this most aggressive type of brain cancer, HCMV is believed to be oncomodulatory. HCMV encodes four viral GPCRs, human chemokine receptor homologs, of which US28 is the best-studied one. This receptor is vital for the establishment and maintenance of latency as well as the oncomodulatory effects. Yet, there are outstanding questions regarding US28 conformations and mechanisms underlying the oncomodulatory effects. Nanobodies or VHHs are the single variable fragments of heavy chain-only antibodies found in Camelidae family members (e.g., camels, llamas, alpacas). The long complementarity determining region 3 and protruding paratope make nanobodies ideal for targeting discontinuous cryptic epitopes, such as the binding pockets of GPCRs. This thesis describes the development of nanobodies to study the mechanisms underlying oncogenic signaling by human chemokine receptor CXCR4 and viral chemokine receptor US28. The first study introduces fluorescently labeled nanobodies as probes for bioluminescence resonance energy transfer (BRET)-based binding studies with Nanoluciferasetagged GPCRs. This workflow allows for the real-time monitoring of receptor-ligand interactions at multiple receptor sites, providing insights into ligand binding behaviors. In the second study, we report on VUN103, a nanobody that targets the intracellular site of US28. A comparison of VUN103 with a previously published US28-targeting nanobody, Nb7, demonstrates the existence of different active conformations. While VUN103 recognizes the apo constitutively active state of US28, Nb7 recognizes the ligand-bound active state. By interfering with G protein binding, VUN103 could fully inhibit US28 signaling and associated oncomodulatory effects in glioblastoma cells. The third study sheds light on how US28 rewires and relocalizes cellular signaling networks in glioblastoma cells, thereby contributing to HCMV-mediated oncomodulatory effects. US28 activates the sphingosine kinase-1 (SK1)/sphingosine-1-phosphate receptor 1 (S1P1) signaling axis, apparently from intracellular sites, thereby engaging in malignant feed-forward loops that exert proliferative and anti-apoptotic effects. Abstract 8 Overall, the research described in this thesis provides insights into the mechanisms underlying the oncogenic signaling of human and viral chemokine receptors. The existence and functional relevance of GPCR oligomers and multiple active conformations, as well as malignant intracellular signaling complexes, are key findings. Furthermore, this thesis highlights the versatility of nanobodies to study and modulate various aspects of GPCR interactions and signaling.