Fungal photoinactivation doses for UV radiation and visible light-a data collection.

Nearly two million people die each year from fungal infections. Additionally, fungal crop infections jeopardize the global food supply. The use of 254 nm UVC radiation from mercury vapor lamps is a disinfection technique known to be effective against all microorganisms, and there are surveys of published UVC sensitivities. However, these mainly focus on bacteria and viruses. Therefore, a corresponding overview for fungi will be provided here, including far-UVC, UVB, UVA, and visible light, in addition to the conventional 254 nm UVC inactivation. The available literature was searched for photoinactivation data for fungi in the above-mentioned spectral ranges. To standardize the presentation, the mean log-reduction doses were retrieved and sorted by fungal species, spectral range, wavelength, and medium, among others. Additionally, the median log-reduction dose was determined for fungi in transparent liquid media. Approximately 400 evaluable individual data sets from publications over the last 100 years were compiled. Most studies were performed with 254 nm radiation from mercury vapor lamps on Aspergillus niger, Candida albicans, and Saccharomyces cerevisiae. However, the data found were highly scattered, which could be due to the experimental conditions. Even though the number of individual data sets seems large, many important fungi have not been extensively studied so far. For example, UV irradiation data does not yet exist for half of the fungal species classified as "high priority" or "medium priority" by the World Health Organization (WHO). In addition, researchers should measure the transmission of their fungal suspensions at the irradiation wavelength to avoid the undesirable effects of either absorption or scattering on irradiation results.

Photoinactivation of the bacteriophage PhiX174 by UVA radiation and visible light in SM buffer and DMEM-F12.

OBJECTIVE: It has been observed that viruses can be inactivated by UVA radiation and visible light. The aim of this study is to investigate whether a medium that contains a photosensitizer might have an influence on viral reduction under irradiation by UVA, violet or blue light. Test virus is the bacteriophage PhiX174 in the photosensitizer-free SM buffer and DMEM-F12, which contains the known photosensitizer riboflavin. RESULTS: The determined PhiX174 D90 doses in SM buffer and DMEM were 36.8 J/cm² and 13.6 J/cm² at 366 nm, 153.6 J/cm² and 129.1 J/cm² at 408 nm and 4988 J/cm² and 2477.1 J/cm² at 455 nm, respectively. It can be concluded that the medium has a large influence on the results. This might be caused by the photosensitizer riboflavin in DMEM-F12. As riboflavin is a key component in many cell culture media, irradiation experiments with viruses in cell culture media should be avoided if the investigation of intrinsical photoinactivation properties of viruses is aimed for.

UV radiation sensitivity of bacteriophage PhiX174 - A potential surrogate for SARS-CoV-2 in terms of radiation inactivation.

To minimize health risks, surrogates are often employed to reduce experiments with pathogenic microorganisms and the associated health risk. Due to structural similarities between the enveloped RNA-viruses SARS-CoV-2 and Phi6, the latter has been established as a nonpathogenic coronavirus surrogate for many applications. However, large discrepancies in the UV log-reduction doses between SARS-CoV-2 and Phi6 necessitate the search for a better surrogate for UV inactivation applications. A literature study provided the bacteriophage PhiX174 as a potentially more suitable nonpathogenic coronavirus surrogate candidate. In irradiation experiments, the sensitivity of PhiX174 was investigated upon exposure to UV radiation of wavelengths 222 nm (Far-UVC), 254 nm (UVC), 302 nm (broad-band UVB), 311 nm (narrow-band UVB) and 366 nm (UVA) using a plaque assay. The determined log-reduction doses for PhiX174 were 1.3 mJ/cm2 @ 222 nm, 5 mJ/cm2 @ 254 nm, 17.9 mJ/cm2 @ 302 nm, 625 mJ/cm2 @ 311 nm and 42.5 J/cm2 @ 366 nm. The comparison of these results with published log-reduction doses of SARS-CoV-2 in the same spectral region, led to the conclusion that the bacteriophage PhiX174 exhibits larger log-reduction doses than SARS-CoV-2, nevertheless, it is a better UV-surrogate at 222 nm (Far-UVC), 254 nm (UVC) and 302 nm (UVB) than the often applied Phi6.

Sensitivity of influenza virus to ultraviolet irradiation.

Background: The measures implemented against the coronavirus pandemic also led to a sharp decline in influenza infections in the 2020/2021 flu season. In the meantime, however, the number of influenza infections has risen again; it is known from history that influenza viruses can also trigger severe pandemics. Therefore, we investigated the efficacy of ultraviolet radiation in the spectral range of 200-400 nm for inactivating influenza viruses. Materials and methods: The scientific literature was searched for published ultraviolet (UV) irradiation experiments with influenza viruses and the results were standardized by determining the lg-reduction dose. The results were then sorted and analyzed by virus type and wavelength as far as possible. Results: The scope of the published data sets was limited and revealed large variations with regard to the lg-reduction dose. Only for experiments with influenza viruses in liquid media in the UVC spectral range around 260 nm - the emission range of commonly-used mercury vapor lamps - was there sufficient data to compare virus types. No significant difference between the virus (sub-) types was observed. The lg-reduction dose in this spectral range is 1.75 mJ/cm2 (median). It was also shown that influenza viruses are particularly sensitive in the far-UVC spectral range (200-230 nm). Conclusion: UVC, including far-UVC, is suited for influenza virus inactivation as long as the viruses are in UVC-transparent materials. A large difference in the UV sensitivity of different influenza viruses from the last approx. 100 years could not be detected. Thus, it is reasonable to assume that future influenza viruses will also be similarly UV-sensitive or that UV can also inactivate new influenza viruses.

UVC, UVB and UVA susceptibility of Phi6 and its suitability as a SARS-CoV-2 surrogate.

For SARS-CoV-2 disinfection systems or applications that are based on UVC, UVB or UVA irradiation, it would be desirable to have a SARS-CoV-2 surrogate for tests and development, which does not require a laboratory with a high biosafety level. The bacteriophage Phi 6, an enveloped RNA virus like coronaviruses, is an obvious candidate for such a surrogate. In this study, UVC, UVB and UVA log-reduction doses for Phi6 are determined by plaque assay. Log-reduction doses for SARS-CoV-2 are retrieved from a literature research. Because of a high variability of the published results, median log-reduction doses are determined for defined spectral ranges and compared to Phi6 data in the same intervals. The measured Phi6 log-reduction doses for UVC (254 nm), UVB (311 nm) and UVA (365 nm) are 31.7, 980 and 14 684 mJ/cm2, respectively. The determined median log-reduction doses for SARS-CoV-2 are much lower, only about 1.7 mJ/cm2 within the spectral interval 251-270 nm. Therefore, Phi6 can be photoinactivated by all UV wavelengths but it is much less UV sensitive compared to SARS-CoV-2 in all UV spectral ranges. Thus, Phi6 is no convincing SARS-CoV-2 surrogate in UV applications.

Blue light inactivation of the enveloped RNA virus Phi6.

OBJECTIVE: Ultraviolet radiation is known for its antimicrobial properties but unfortunately, it could also harm humans. Currently, disinfection techniques against SARS-CoV-2 are being sought that can be applied on air and surfaces and which do not pose a relevant thread to humans. In this study, the bacteriophage phi6, which like SARS-CoV-2 is an enveloped RNA virus, is irradiated with visible blue light at a wavelength of 455 nm. RESULTS: For the first time worldwide, the antiviral properties of blue light around 455 nm can be demonstrated. With a dose of 7200 J/cm2, the concentration of this enveloped RNA virus can be successfully reduced by more than three orders of magnitude. The inactivation mechanism is still unknown, but the sensitivity ratio of phi6 towards blue and violet light hints towards an involvement of photosensitizers of the host cells. Own studies on coronaviruses cannot be executed, but the results support speculations about blue-susceptibility of coronaviruses, which might allow to employ blue light for infection prevention or even therapeutic applications.

The impact of far-UVC radiation (200-230 nm) on pathogens, cells, skin, and eyes - a collection and analysis of a hundred years of data.

Background: The ongoing coronavirus pandemic requires new disinfection approaches, especially for airborne viruses. The 254 nm emission of low-pressure vacuum lamps is known for its antimicrobial effect, but unfortunately, this radiation is also harmful to human cells. Some researchers published reports that short-wavelength ultraviolet light in the spectral region of 200-230 nm (far-UVC) should inactivate pathogens without harming human cells, which might be very helpful in many applications. Methods: A literature search on the impact of far-UVC radiation on pathogens, cells, skin and eyes was performed and median log-reduction doses for different pathogens and wavelengths were calculated. Observed damage to cells, skin and eyes was collected and presented in standardized form. Results: More than 100 papers on far-UVC disinfection, published within the last 100 years, were found. Far-UVC radiation, especially the 222 nm emission of KrCl excimer lamps, exhibits strong antimicrobial properties. The average necessary log-reduction doses are 1.3 times higher than with 254 nm irradiation. A dose of 100 mJ/cm2 reduces all pathogens by several orders of magnitude without harming human cells, if optical filters block emissions above 230 nm. Conclusion: The approach is very promising, especially for temporary applications, but the data is still sparse. Investigations with high far-UVC doses over a longer period of time have not yet been carried out, and there is no positive study on the impact of this radiation on human eyes. Additionally, far-UVC sources are unavailable in larger quantities. Therefore, this is not a short-term solution for the current pandemic, but may be suitable for future technological approaches for decontamination in rooms in the presence of people or for antisepsis.

Photoinactivation of the Coronavirus Surrogate phi6 by Visible Light.

To stop the coronavirus spread, new inactivation approaches are being sought that can also be applied in the presence of humans or even on humans. Here, we investigate the effect of visible violet light with a wavelength of 405 nm on the coronavirus surrogate phi6 in two aqueous solutions that are free of photosensitizers. A dose of 1300 J cm-2 of 405 nm irradiation reduces the phi6 plaque-forming unit concentration by three log-levels. The next step should be similar visible light photoinactivation investigations on coronaviruses, which cannot be performed in our lab.

Ultraviolet irradiation doses for coronavirus inactivation - review and analysis of coronavirus photoinactivation studies.

Background: To slow the increasing global spread of the SARS-CoV-2 virus, appropriate disinfection techniques are required. Ultraviolet radiation (UV) has a well-known antiviral effect, but measurements on the radiation dose necessary to inactivate SARS-CoV-2 have not been published so far. Methods: Coronavirus inactivation experiments with ultraviolet light performed in the past were evaluated to determine the UV radiation dose required for a 90% virus reduction. This analysis is based on the fact that all coronaviruses have a similar structure and similar RNA strand length. Results: The available data reveals large variations, which are apparently not caused by the coronaviruses but by the experimental conditions selected. If these are excluded as far as possible, it appears that coronaviruses are very UV sensitive. The upper limit determined for the log-reduction dose (90% reduction) is approximately 10.6 mJ/cm2 (median), while the true value is probably only 3.7 mJ/cm2 (median). Conclusion: Since coronaviruses do not differ structurally to any great exent, the SARS-CoV-2 virus - as well as possible future mutations - will very likely be highly UV sensitive, so that common UV disinfection procedures will inactivate the new SARS-CoV-2 virus without any further modification.

Antimicrobial Effect of Visible Light-Photoinactivation of Legionella rubrilucens by Irradiation at 450, 470, and 620 nm.

Despite the high number of legionella infections, there are currently no convincing preventive measures. Photoinactivation with visible light is a promising new approach and the photoinactivation sensitivity properties of planktonic Legionella rubrilucens to 450, 470, and 620 nm irradiation were thus investigated and compared to existing 405 nm inactivation data for obtaining information on responsible endogenous photosensitizers. Legionella were streaked on agar plates and irradiated with different doses by light emitting diodes (LEDs) of different visible wavelengths. When irradiating bacterial samples with blue light of 450 nm, a 5-log reduction could be achieved by applying a dose of 300 J cm-2, whereas at 470 nm, a comparable reduction required about 500 J cm-2. For red irradiation at 620 nm, no inactivation could be observed, even at 500 J cm-2. The declining photoinactivation sensitivity with an increasing wavelength is consistent with the assumption of porphyrins and flavins being among the relevant photosensitizers. These results were obtained for L. rubrilucens, but there is reason to believe that its inactivation behavior is similar to that of pathogenic legionella species. Therefore, this photoinactivation might lead to new future concepts for legionella reduction and prevention in technical applications or even on or inside the human body.

UV-C inactivation of Legionella rubrilucens.

Background: Despite the great health significance of Legionella, there is only little information on their UV sensitivity. Besides Legionella pneumophila only L. longbeachae has been investigated so far. Methods: In this study L. rubrilucens has been spread on buffered charcoal yeast extract agar and irradiated with the 254 nm UV-C emission of a mercury vapor lamp. The disinfection success is measured by colony counting after incubation and comparison of the number of colonies on irradiated and unirradiated reference agar plates. Results: The average log-reduction dose is 1.08 mJ/cm2 for free L. rubrilucens, which is at the lower end of the so far published Legionella log-reduction values, but all three Legionella species show similar UV-C sensitivities. Conclusion: The log-reduction dose of legionellae in amoebae has not been investigated, but with the observed high UV-C sensitivity for free Legionella, the idea of a future point-of-use disinfection by small UV-C LEDs in water-taps or shower heads appears to be realistic, even if legionellae are more resistant in amoebae.

Cool-temperature-mediated activation of phospholipase C-γ2 in the human hereditary disease PLAID.

Deletions in the gene encoding signal-transducing inositol phospholipid-specific phospholipase C-γ2 (PLCγ2) are associated with the novel human hereditary disease PLAID (PLCγ2-associated antibody deficiency and immune dysregulation). PLAID is characterized by a rather puzzling concurrence of augmented and diminished functions of the immune system, such as cold urticaria triggered by only minimal decreases in temperature, autoimmunity, and immunodeficiency. Understanding of the functional effects of the genomic alterations at the level of the affected enzyme, PLCγ2, is currently lacking. PLCγ2 is critically involved in coupling various cell surface receptors to regulation of important functions of immune cells such as mast cells, B cells, monocytes/macrophages, and neutrophils. PLCγ2 is unique by carrying three Src (SH) and one split pleckstrin homology domain (spPH) between the two catalytic subdomains (spPHn-SH2n-SH2c-SH3-spPHc). Prevailing evidence suggests that activation of PLCγ2 is primarily due to loss of SH-region-mediated autoinhibition and/or enhanced plasma membrane translocation. Here, we show that the two PLAID PLCγ2 mutants lacking portions of the SH region are strongly (>100-fold), rapidly, and reversibly activated by cooling by only a few degrees. We found that the mechanism(s) underlying PLCγ2 PLAID mutant activation by cool temperatures is distinct from a mere loss of SH-region-mediated autoinhibition and dependent on both the integrity and the pliability of the spPH domain. The results suggest a new mechanism of PLCγ activation with unique thermodynamic features and assign a novel regulatory role to its spPH domain. Involvement of this mechanism in other human disease states associated with cooling such as exertional asthma and certain acute coronary events appears an intriguing possibility.

Ligand-dependent serum response factor activation by the human CC chemokine receptors CCR2a and CCR2b is mediated by G proteins of the Gq family.

Expression of the human CCR2 receptors, CCR2a and CCR2b, in mammalian cells results in ligand-dependent changes in the activity of multiple cellular signal transduction pathways, mediated in most cases by pertussis toxin-sensitive heterotrimeric G proteins of the Gi/o subfamily. In addition, CCR2a and CCR2b receptors have been shown to couple to Gq family members, triggering the canonical activation of phospholipase Cß isoenzymes. Activation of pertussis toxin-insensitive Gq proteins by cell-surface receptors is not only coupled to activation of phospholipase isoenzymes but also to Rho guanine nucleotide exchange factors, which in turn mediate activation of the Rho GTPases. Activated Rho GTPases regulate numerous cellular functions, including the organization of the actin cytoskeleton and gene transcription, such as the transcription factor serum response factor. These findings prompted us to investigate whether CCR2a and/or CCR2b stimulate serum response factor activity. The results presented herein demonstrate that stimulation of human CCR2a- or CCR2b-expressing COS-7 cells caused a vigorous induction of serum response factor activity. This effect was specifically mediated by Gq and/or G14, as well as Rho A and/or a closely related Rho GTPase. Furthermore, the stimulatory effect of CCR2a and CCR2b and Gαq was sensitive to coexpression of the Gαq-interacting leukemia-associated Rho guanine nucleotide exchange factor. The findings of the work indicate a role for Gαq and/or Gα14 and in CCR2a/CCR2b-stimulated Rho A GTPase-mediated serum response factor activation and introduce a noncanonical pathway activated by CCR2 receptors by coupling to Gq proteins.

Biologically inactive leptin and early-onset extreme obesity.

Mutations in the gene encoding leptin (LEP) typically lead to an absence of circulating leptin and to extreme obesity. We describe a 2-year-old boy with early-onset extreme obesity due to a novel homozygous transversion (c.298G→T) in LEP, leading to a change from aspartic acid to tyrosine at amino acid position 100 (p.D100Y) and high immunoreactive levels of leptin. Overexpression studies confirmed that the mutant protein is secreted but neither binds to nor activates the leptin receptor. The mutant protein failed to reduce food intake and body weight in leptin-deficient ob/ob mice. Treatment of the patient with recombinant human leptin (metreleptin) rapidly normalized eating behavior and resulted in weight loss.

LARG links histamine-H1-receptor-activated Gq to Rho-GTPase-dependent signaling pathways.

Activation of heterotrimeric G proteins, such as G(12/13) and G(q), by cell surface receptors is coupled to the regulation of numerous cellular functions controlled by activated Rho GTPases. Previous studies have implicated the Rho guanine nucleotide exchange factor (RhoGEF) leukemia-associated RhoGEF (LARG) as a regulatory protein receiving stimulatory inputs from activated Gα(12/13) and Gα(q). However, the molecular mechanisms of the Gα(q)-mediated LARG activation are not fully understood and the structural elements of LARG involved in this process have remained unclear. In the present work, the specific coupling of the histamine H1 receptor (HRH1) exogenously expressed in COS-7 cells to G(q), but not to G(12/13), was used to conduct a detailed analysis of receptor- and Gα(q)-mediated LARG activation and to define its structural requirements. The results show that HRH1-mediated activation of the strictly Rho-dependent transcriptional activity of serum response factor requires the PDZ domain of LARG and can be mimicked by activated Gα(q)(Q209L). The functional interaction between activated Gα(q) and LARG requires no more than the catalytic DH-PH tandem of LARG, and is independent of PLCß activation and distinct from the mechanisms of Gα(q)-mediated p63RhoGEF and PLCß(3) activation. Activated Gα(q) physically interacts with the relevant portions of LARG in COS-7 cells and histamine causes activation of LARG in native HeLa cells endogenously expressing HRH1, G(q), and LARG. This work is the first positive demonstration of a stimulatory effect of LARG on the ability of a strictly G(q)-coupled receptor to cause activation of a Rho-GTPase-dependent signaling pathway.

Membrane environment exerts an important influence on rac-mediated activation of phospholipase Cγ2.

We performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation.

Structural insights into formation of an active signaling complex between Rac and phospholipase C gamma 2.

Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-gamma(2) (PLCgamma(2)). Based on this structure, we illustrate distinct requirements for PLCgamma(2) activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLCgamma(2), but not the related PLCbeta(2) isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTPgammaS. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors.

rac regulates its effector phospholipase Cgamma2 through interaction with a split pleckstrin homology domain.

Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLCgamma isoforms, only PLCgamma(2) has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLCgamma(2) required for activation. Based on reconstitution experiments with isolated PLCgamma variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLCgamma(2) by Rac2. We also demonstrate that Rac2 directly binds to PLCgamma(2) as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLCgamma(1) and PLCgamma(2) and the implications of our findings for their respective signaling roles.

Constitutive serum response factor activation by the viral chemokine receptor homologue pUS28 is differentially regulated by Galpha(q/11) and Galpha(16).

Expression of the human cytomegalovirus (HCMV)-encoded chemokine receptor homologue pUS28 in mammalian cells results in ligand-dependent and -independent changes in the activity of multiple cellular signal transduction pathways. The ligand-dependent signalling activity of pUS28 has been shown to be predominantly mediated by heterotrimeric G proteins of the G(i/o) and G(12/13) subfamilies. Ligand-independent constitutive activity of pUS28 causing stimulation of inositol phosphate formation has been correlated with the coupling of pUS28 to G proteins of the G(q) family. It is well known that activation of G(q) proteins by cell surface receptors is coupled to activation of the Rho GTPase RhoA. Activated RhoA regulates numerous cellular functions, including the activity of the transcription factor serum response factor (SRF). The marked activation of G(q) proteins by pUS28 in transfected and HCMV-infected cells prompted us to investigate its effect on SRF activity. The results presented herein demonstrate that expression of pUS28 in COS-7 cells caused a vigorous induction of SRF activity. This effect was observed in the absence of chemokines known to interact with pUS28, and was specifically mediated by endogenous G(q) and/or G(11) as well as RhoA and/or a closely related Rho GTPase. The stimulatory effect of pUS28 and Galpha(q/11) was independent of phospholipase C-beta (PLCbeta) activation and was markedly sensitive to inhibition by wild-type, but not by constitutively active Galpha(16), thus identifying Galpha(16) as a modulator of Galpha(q/11) function likely to act by competing with Galpha(q/11) for and thus uncoupling Galpha(q/11) from activation by pUS28.

The variable C-terminal extension of G-protein-coupled receptor kinase 6 constitutes an accessorial autoregulatory domain.

G-protein-coupled receptor kinases (GRK) are known to phosphorylate agonist-occupied G-protein-coupled receptors. We expressed and functionally characterized mouse GRK6 proteins encoded by four distinct mRNAs generated by alternative RNA splicing from a single gene, mGRK6-A to mGRK6-D. Three isoforms, mGRK6-A to mGRK6-C differ in their C-terminal-most portion, which is known to mediate membrane and/or receptor interaction and regulate the activity of GRK4-like kinases. One isoform, mGRK6-D, is identical to the other mGRK6 variants in the N-terminal region, but carries an incomplete catalytical domain. Mouse GRK6-D was catalytically inactive and specifically present in the nucleus of transfected cells. Recombinant mouse GRK6-A to mGRK6-C were found to be membrane-associated in cell-free systems and in transfected COS-7 cells, suggesting that the very C-terminus of GRK6-A, lacking in GRK6-B and mGRK6-C and carrying consensus sites for palmitoylation, is not required for membrane interaction. Interestingly, the shortest catalytically active variant, mGRK6-C, was conspicuously more active in phosphorylating light-activated rhodopsin than mGRK6-A and mGRK6-B, implying that the C-terminus of the latter two variants may fulfil an autoinhibitory function. Mutation and removal of C-terminal-most region of mGRK6-A by site-directed mutagenesis revealed that this region contains three autoregulatory elements: two discontinuous inhibitory elements consisting of a single residue, D560, and the sequence between residues S566 and L576, and an intervening stimulatory element. The results suggest that mGRK6-C may be considered a basic, prototypic representative of the GRK4-like kinases, which is capable of interacting with both plasma membrane and its receptor substrate, but is resistant to further regulatory modification conferred to the prototype via C-terminal extension.