RESUMO
Thermal lens spectrometry along with spectrophotometric titration were used to assess the composition of the complex of oxidized cytochrome c (ferricytochrome c) with 1,1',2,2'-tetraoleyl cardiolipin, which plays a key role in the initiation of apoptosis. Spectrophotometric titration was carried out for micromolar concentrations at which the complex is mainly insoluble, to assess the residual concentration in the solution and to estimate the solubility of the complex. Thermal lens spectrometry was used as a method of molecular absorption spectroscopy, which has two advantages over conventional optical transmission spectroscopy: the higher sensitivity of absorbance measurements and the possibility of studying the light absorption by chromophores and heat transfer in complex systems, such as living cells or tissues. Thermal lens measurements were carried out at nanomolar concentrations, where the complex is mainly in solution, i.e., under the conditions of its direct measurements. From the thermal lens measurements, the ratios of cytochrome c and cardiolipin in the complex were 50 at pH 7.4; 30 at pH 6.8; and 10 at pH 5.5, which fit well to the spectrophotometric data. The molecular solubility of the complex at pH 6.8-7.4 was estimated as 30 µmol/L.
Assuntos
Cardiolipinas , Citocromos c , Citocromos c/química , Cardiolipinas/química , EspectrofotometriaRESUMO
T cells become dysfunctional when they encounter self antigens or are exposed to chronic infection or to the tumour microenvironment1. The function of T cells is tightly regulated by a combinational co-stimulatory signal, and dominance of negative co-stimulation results in T cell dysfunction2. However, the molecular mechanisms that underlie this dysfunction remain unclear. Here, using an in vitro T cell tolerance induction system in mice, we characterize genome-wide epigenetic and gene expression features in tolerant T cells, and show that they are distinct from effector and regulatory T cells. Notably, the transcription factor NR4A1 is stably expressed at high levels in tolerant T cells. Overexpression of NR4A1 inhibits effector T cell differentiation, whereas deletion of NR4A1 overcomes T cell tolerance and exaggerates effector function, as well as enhancing immunity against tumour and chronic virus. Mechanistically, NR4A1 is preferentially recruited to binding sites of the transcription factor AP-1, where it represses effector-gene expression by inhibiting AP-1 function. NR4A1 binding also promotes acetylation of histone 3 at lysine 27 (H3K27ac), leading to activation of tolerance-related genes. This study thus identifies NR4A1 as a key general regulator in the induction of T cell dysfunction, and a potential target for tumour immunotherapy.
Assuntos
Regulação da Expressão Gênica/genética , Genoma , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologia , Acetilação , Animais , Infecções por Arenaviridae/imunologia , Infecções por Arenaviridae/virologia , Linhagem Celular Tumoral , Colite/imunologia , Colite/patologia , Colite/terapia , Epigênese Genética , Feminino , Histonas/química , Histonas/metabolismo , Tolerância Imunológica/genética , Imunoterapia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Linfócitos T/imunologia , Fator de Transcrição AP-1/metabolismo , Transcrição GênicaRESUMO
Somatic KRAS mutations are the most common oncogenic variants in lung cancer and are associated with poor prognosis. Using a Kras-induced lung cancer mouse model, CC-LR, we previously showed a role for inflammation in lung tumorigenesis through activation of the NF-κB pathway, along with induction of interleukin 6 (IL6) and an IL17-producing CD4+ T-helper cell response. IL22 is an effector molecule secreted by CD4+ and γδ T cells that we previously found to be expressed in CC-LR mice. IL22 mostly signals through the STAT3 pathway and is thought to act exclusively on nonhematopoietic cells with basal IL22 receptor (IL22R) expression on epithelial cells. Here, we found that higher expression of IL22R1 in patients with KRAS-mutant lung adenocarcinoma was an independent indicator of poor recurrence-free survival. We then showed that genetic ablation of Il22 in CC-LR mice (CC-LR/IL22KO mice) caused a significant reduction in tumor number and size. This was accompanied by significantly lower tumor cell proliferation, angiogenesis, and STAT3 activation. Il22 ablation was also associated with significant reduction in lung-infiltrating inflammatory cells and expression of protumor inflammatory cytokines. Conversely, this was accompanied with increased antitumor Th1 and cytotoxic CD8+ T-cell responses, while suppressing the protumor immunosuppressive T regulatory cell response. In CC-LR/IL22KO mice, we found significantly reduced expression of core stemness genes and the number of prototypical SPC+CCSP+ stem cells. Thus, we conclude that IL22 promotes Kras-mutant lung tumorigenesis by driving a protumor inflammatory microenvironment with proliferative, angiogenic, and stemness contextual cues in epithelial/tumor cells. Cancer Immunol Res; 6(7); 788-97. ©2018 AACR.
Assuntos
Interleucinas/metabolismo , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Mutação , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Animais Geneticamente Modificados , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Humanos , Imuno-Histoquímica , Interleucinas/genética , Neoplasias Pulmonares/patologia , Camundongos , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Microambiente Tumoral , Interleucina 22RESUMO
T-cell tolerance is a major obstacle to successful cancer immunotherapy; thus, developing strategies to break immune tolerance is a high priority. Here we show that expression of the E3 ubiquitin ligase Grail is upregulated in CD8+ T cells that have infiltrated into transplanted lymphoma tumours, and Grail deficiency confers long-term tumour control. Importantly, therapeutic transfer of Grail-deficient CD8+ T cells is sufficient to repress established tumours. Mechanistically, loss of Grail enhances anti-tumour reactivity and functionality of CD8+ T cells. In addition, Grail-deficient CD8+ T cells have increased IL-21 receptor (IL-21R) expression and hyperresponsiveness to IL-21 signalling as Grail promotes IL-21R ubiquitination and degradation. Moreover, CD8+ T cells isolated from lymphoma patients express higher levels of Grail and lower levels of IL-21R, compared with CD8+ T cells from normal donors. Our data demonstrate that Grail is a crucial factor controlling CD8+ T-cell function and is a potential target to improve cytotoxic T-cell activity.Grail is an E3 ubiquitin ligase that inhibits T-cell receptor signalling in CD4+ T cells. Here the authors show Grail also limits IL-21 receptor expression and function in CD8+ T cells, is overactive in these cells in patients with lymphoma, and promotes tumour development in a lymphoma transplant mouse model.
Assuntos
Linfócitos T CD8-Positivos/enzimologia , Linfoma/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Humanos , Tolerância Imunológica , Interleucinas/genética , Interleucinas/imunologia , Linfoma/enzimologia , Linfoma/genética , Camundongos , Camundongos Knockout , Infiltração de Neutrófilos , Receptores de Interleucina-21/genética , Receptores de Interleucina-21/imunologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
T follicular helper (Tfh) cells are specialized subset of T helper (Th) cells necessary for germinal center reaction, affinity maturation and the differentiation of germinal center B cells to antibody-producing plasma B cells and memory B cells. The differentiation of Tfh cells is a multistage, multifactorial process involving a variety of cytokines, surface molecules and transcription factors. While Tfh cells are critical components of protective immune responses against pathogens, regulation of these cells is crucial to prevent autoimmunity and airway inflammation. Recently, it has been noted that Tfh cells could be potentially implicated either in cancer progression or prevention. Thus, the elucidation of the mechanisms that regulate Tfh cell differentiation, function and fate should highlight potential targets for novel therapeutic approaches. In this review, we summarize the latest advances in our understanding of the regulation of Tfh cell differentiation and their role in health and disease.
Assuntos
Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/fisiologia , Animais , Autoimunidade , Linfócitos B/imunologia , Diferenciação Celular , Citocinas/imunologia , Regulação da Expressão Gênica , Centro Germinativo/citologia , Humanos , Memória Imunológica , Interleucina-4/biossíntese , Interleucina-4/imunologia , Ativação Linfocitária , Camundongos , Células Th2/imunologiaRESUMO
Apart from T helper (Th)-2 cells, T follicular helper (Tfh) cells are a major class of IL-4-producing T cells, required for regulation of type 2 humoral immunity; however, transcriptional control of IL-4 production in Tfh cells remains mainly unknown. Here, we show that the basic leucine zipper transcription factor ATF-like, Batf is important for IL-4 expression in Tfh cells rather than in canonical Th2 cells. Functionally, Batf in cooperation with interferon regulatory factor (IRF) 4 along with Stat3 and Stat6 trigger IL-4 production in Tfh cells by directly binding to and activation of the CNS2 region in the IL-4 locus. In addition, Batf-to-c-Maf signalling is an important determinant of IL-4 expression in Tfh cells. Batf deficiency impairs the generation of IL-4-producing Tfh cells that results in protection against allergic asthma. Our results thus indicate a positive role of Batf in promoting the generation of pro-allergic IL-4-producing Tfh cells.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação da Expressão Gênica/fisiologia , Interleucina-4/metabolismo , Linfócitos T Auxiliares-Indutores/fisiologia , Transferência Adotiva , Animais , Asma/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Células da Medula Óssea , Diferenciação Celular , Imunoprecipitação da Cromatina , Interleucina-4/genética , Masculino , Camundongos , Camundongos Knockout , Ovalbumina/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismoRESUMO
T helper (Th)-2 cells are the major players in allergic asthma; however, the mechanisms that control Th2-mediated inflammation are poorly understood. Here we find that enhanced expression of Grail, an E3 ubiquitin ligase, in Th2 cells depends on interleukin (IL)-4-signalling components, signal transducer and activator of transcription 6 (Stat6) and Gata3, that bind to and transactivate the Grail promoter. Grail deficiency in T cells leads to increased expression of Th2 effector cytokines in vitro and in vivo and Grail-deficient mice are more susceptible to allergic asthma. Mechanistically, the enhanced effector function of Grail-deficient Th2 cells is mediated by increased expression of Stat6 and IL-4 receptor α-chain. Grail interacts with Stat6 and targets it for ubiquitination and degradation. Thus, our results indicate that Grail plays a critical role in controlling Th2 development through a negative feedback loop.
Assuntos
Asma/imunologia , Fator de Transcrição STAT6/metabolismo , Células Th2/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Asma/genética , Asma/patologia , Diferenciação Celular , Retroalimentação Fisiológica , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Células HEK293 , Humanos , Interleucina-4/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Th2/efeitos dos fármacos , Células Th2/fisiologia , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation is mediated by the activation of recipient dendritic cells and subsequent proliferation of donor T cells. The complement system was recently shown to modulate adaptive immunity through an interaction of the complement system and lymphocytes. Complement proteins participate in the activation of dendritic cells, antigen presentation to T cells, and proliferation of T cells. Our studies with a murine model of bone marrow transplantation demonstrate that complement system regulates alloimmune responses in GVHD. Mice deficient in the central component of the complement system (C3(-/-)) had significantly lower GVHD-related mortality and morbidity compared with wild-type recipient mice. The numbers of donor-derived T cells, including IFN-γ(+), IL-17(+), and IL-17(+)IFN-γ(+) subsets, were decreased in secondary lymphoid organs of C3(-/-) recipients. Furthermore, the number of recipient CD8α(+)CD11c(+) cells in lymphoid organs was reduced. We conclude that C3 regulates Th1/17 differentiation in bone marrow transplantation, and define a novel function of the complement system in GVHD.
Assuntos
Complemento C3/deficiência , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Células Th1/imunologia , Células Th17/imunologia , Animais , Diferenciação Celular/imunologia , Complemento C3/imunologia , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Camundongos , Camundongos Endogâmicos BALB C , Células Th1/patologia , Células Th17/patologia , Quimeras de Transplante , Transplante HomólogoRESUMO
Recent work has identified a new subset of CD4(+) T cells named as Tfh cells that are localized in germinal centers and critical in germinal center formation. Tfh cell differentiation is regulated by IL-6 and IL-21, possibly via STAT3 factor, and B cell lymphoma 6 (Bcl6) is specifically expressed in Tfh cells and required for their lineage specification. In the current study, we characterized the role of STAT5 in Tfh cell development. We found that a constitutively active form of STAT5 effectively inhibited Tfh differentiation by suppressing the expression of Tfh-associated factors (CXC motif) receptor 5 (CXCR5), musculoaponeurotic fibrosarcoma (c-Maf), Bcl6, basic leucine zipper transcription factor ATF-like (Batf), and IL-21, and STAT5 deficiency greatly enhanced Tfh gene expression. Importantly, STAT5 regulated the expression of Tfh cell suppressor factor B lymphocyte-induced maturation protein 1 (Blimp-1); STAT5 deficiency impaired Blimp-1 expression and resulted in elevated expression of Tfh-specific genes. Similarly, inhibition of IL-2 potentiated Tfh generation, associated with dampened Blimp-1 expression; Blimp-1 overexpression inhibited Tfh gene expression in Stat5-deficient T cells, suggesting that the IL-2/STAT5 axis functions to regulate Blimp-1 expression. In vivo, deletion of STAT5 in CD4(+) T cells resulted in enhanced development of Tfh cells and germinal center B cells and led to an impairment of B cell tolerance in a well defined mouse tolerance model. Taken together, this study demonstrates that STAT5 controls Tfh differentiation.
Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Fator de Transcrição STAT5/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Camundongos , Fator 1 de Ligação ao Domínio I Regulador Positivo , Fator de Transcrição STAT5/deficiência , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Retinitis pigmentosa (RP) is a heterogeneous group of hereditary disorders of the retina caused by mutation in genes of the photoreceptor proteins with an autosomal dominant (adRP), autosomal recessive (arRP), or X-linked pattern of inheritance. Although there are over 100 identified mutations in the opsin gene associated with RP, only a few of them are inherited with the arRP pattern. E150K is the first reported missense mutation associated with arRP. This opsin mutation is located in the second cytoplasmic loop of this G protein-coupled receptor. E150K opsin expressed in HEK293 cells and reconstituted with 11-cis-retinal displayed an absorption spectrum similar to the wild type (WT) counterpart and activated G protein transducin slightly faster than WT receptor. However, the majority of E150K opsin showed a higher apparent molecular mass in SDS-PAGE and was resistant to endoglycosidase H deglycosidase. Instead of being transported to the plasma membrane, E150K opsin is partially colocalized with the cis/medial Golgi compartment markers such as GM130 and Vti1b but not with the trans-Golgi network. In contrast to the endoplasmic reticulum-retained adRP mutant, P23H opsin, Golgi-retained E150K opsin did not influence the proper transport of the WT opsin when coexpressed in HEK293 cells. This result is consistent with the recessive pattern of inheritance of this mutation. Thus, our study reveals a novel molecular mechanism for retinal degeneration that results from deficient export of opsin from the Golgi apparatus.
Assuntos
Mutação de Sentido Incorreto , Retinose Pigmentar/genética , Opsinas de Bastonetes/genética , Linhagem Celular , Genes Recessivos , Complexo de Golgi/metabolismo , Humanos , Transporte Proteico/genética , Degeneração Retiniana/etiologia , Retinaldeído , TransfecçãoRESUMO
The retinoid cycle is a recycling system that replenishes the 11-cis-retinal chromophore of rhodopsin and cone pigments. Photoreceptor-specific retinol dehydrogenase (prRDH) catalyzes reduction of all-trans-retinal to all-trans-retinol and is thought to be a key enzyme in the retinoid cycle. We disrupted mouse prRDH (human gene symbol RDH8) gene expression by targeted recombination and generated a homozygous prRDH knock-out (prRDH-/-) mouse. Histological analysis and electron microscopy of retinas from 6- to 8-week-old prRDH-/- mice revealed no structural differences of the photoreceptors or inner retina. For brief light exposure, absence of prRDH did not affect the rate of 11-cis-retinal regeneration or the decay of Meta II, the activated form of rhodopsin. Absence of prRDH, however, caused significant accumulation of all-trans-retinal following exposure to bright lights and delayed recovery of rod function as measured by electroretinograms and single cell recordings. Retention of all-trans-retinal resulted in slight overproduction of A2E, a condensation product of all-trans-retinal and phosphatidylethanolamine. We conclude that prRDH is an enzyme that catalyzes reduction of all-trans-retinal in the rod outer segment, most noticeably at higher light intensities and prolonged illumination, but is not an essential enzyme of the retinoid cycle.
Assuntos
Oxirredutases do Álcool/fisiologia , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Retinoides/metabolismo , Oxirredutases do Álcool/metabolismo , Animais , Southern Blotting , Catálise , Linhagem Celular , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Eletrorretinografia , Olho/metabolismo , Vetores Genéticos , Genótipo , Humanos , Immunoblotting , Imuno-Histoquímica , Insetos , Cinética , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Modelos Químicos , Modelos Genéticos , Mutação , Fosfatidiletanolaminas/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Recombinação Genética , Retinaldeído/química , Retinoides/química , Rodopsina/química , Rodopsina/metabolismo , Fatores de Tempo , Transgenes , Vitamina A/metabolismoRESUMO
PURPOSE: To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ. METHODS: Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry. RESULTS: A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury. CONCLUSIONS: Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.
Assuntos
Antígeno CD11b/metabolismo , Proteínas de Transporte/metabolismo , Quimiocina CCL2/metabolismo , Substância Própria/metabolismo , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Cicatrização , Animais , Western Blotting , Antígeno CD11b/genética , Proteínas de Transporte/genética , Células Cultivadas , Quimiocina CCL2/genética , Fibroblastos/metabolismo , Glicoproteínas/genética , Proteínas de Fluorescência Verde , Proteínas Luminescentes/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Monócitos/fisiologia , Osteoprotegerina , Testes de Precipitina , Ligante RANK , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B , Receptores Citoplasmáticos e Nucleares/genética , Receptores do Fator de Necrose Tumoral , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Changes in the Ca2+ concentration are thought to affect many processes, including signal transduction in a vast number of biological systems. However, only in few cases the molecular mechanisms by which Ca2+ mediates its action are as well understood as in phototransduction. In dark-adapted photoreceptor cells, the equilibrium level of cGMP is maintained by two opposing activities, such as phosphodiesterase (PDE) and guanylate cyclase (GC). Upon absorption of photons, rhodopsin-G-protein-mediated activation of PDE leads to a transient decrease in [cGMP] and subsequently to lowering of [Ca2+]. In turn, lower [Ca2+] increases net production of cGMP by stimulation of GC until dark conditions are re-established. This activation of GC is mediated by Ca2+ -free forms of Ca2+ -binding proteins termed GC-activating proteins (GCAPs). The last decade brought the molecular identification of GCs and GCAPs in the visual system. Recent efforts have been directed toward understanding the properties of GC at the physiological and structural levels. Here, we summarize the recent progress and present a list of topics of ongoing research.
Assuntos
GMP Cíclico/biossíntese , Variação Genética , Guanilato Ciclase/genética , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Guanilato Ciclase/metabolismo , Proteínas Ativadoras de Guanilato Ciclase , Humanos , Dados de Sequência Molecular , Família Multigênica , Estrutura Terciária de Proteína , Receptores de Superfície Celular/metabolismo , Alinhamento de SequênciaRESUMO
Among single-spanning transmembrane receptors (sTMRs), two guanylyl cyclase receptors, GC1 and GC2, are critically important during phototransduction in vertebrate retinal photoreceptor cells. Ca(2+)-free forms of guanylyl cyclase-activating proteins (GCAPs) stimulate GCs intracellularly by a molecular mechanism that is not fully understood. To gain further insight into the mechanism of activation and specificity among these proteins, for the first time, several soluble and active truncated GCs and fusion proteins between intracellular domains of GCs and full-length GCAPs were generated. The GC activity of myristoylated GCAP--(437-1054)GC displayed typical [Ca(2+)] dependence, and was further enhanced by ATP and inhibited by guanylyl cyclase inhibitor protein (GCIP). The myristoyl group of GCAP1 appeared to be critical for the inhibition of GCs at high [Ca(2+)], even without membranes. In contrast, calmodulin (CaM)--(437-1054)GC1 fusion protein was inactive, but could be stimulated by exogenous GCAP1. In a series of experiments, we showed that the activation of GCs by linked GCAPs involved intra- and intermolecular mechanisms. The catalytically productive GCAP1--(437-1054)GC1 complex can dissociate, allowing binding and stimulation of the GC1 fusion protein by free GCAP1. This suggests that the intramolecular interactions within the fusion protein have low affinity and are mimicking the native system. We present evidence that the mechanism of GC activation by GCAPs involves a dimeric form of GCs, involves direct interaction between GCs and GCAPs, and does not require membrane components. Thus, fusion proteins may provide an important advance for further structural studies of photoreceptor GCs and other sTMRs with and without different forms of regulatory proteins.