Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes

Ligand-activated receptor tyrosine kinases undergo endocytosis and are transported via endosomes to lysosomes for degradation. This "receptor down-regulation" process is crucial to terminate the cell proliferation signals produced by activated receptors. During the process, ubiquitination of the receptors serves as a sorting signal for their trafficking from endosomes to lysosomes. Here, we describe the role of a deubiquitinating enzyme UBPY/USP8 in the down-regulation of epidermal growth factor (EGF) receptor (EGFR). Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF-stimulated cells. Immunopurified UBPY deubiquitinated EGFR in vitro. In EGF-stimulated cells, UBPY underwent ubiquitination and bound to EGFR. Overexpression of Hrs or a dominant-negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes. A catalytically inactive UBPY mutant clearly localized on endosomes, where it overlapped with EGFR when cells were stimulated with EGF. Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF-activated EGFR. We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.     

The RING finger of c-Cbl mediates desensitization of the epidermal growth factor receptor.

Ligand-induced activation of surface receptors, including the epidermal growth factor receptor (EGFR), is followed by a desensitization process involving endocytosis and receptor degradation. c-Cbl, a tyrosine phosphorylation substrate shared by several signaling pathways, accelerates desensitization by recruiting EGFR and increasing receptor polyubiquitination. Here we demonstrate that the RING type zinc finger of c-Cbl is essential for ubiquitination and subsequent desensitization of EGFR. Mutagenesis of a single cysteine residue impaired the ability of c-Cbl to enhance both down-regulation and ubiquitination of EGFR in living cells, although the mutant retained binding to the activated receptor. Consequently, the mutant form of c-Cbl acquired a dominant inhibitory function and lost the ability to inhibit signaling downstream to EGFR. In vitro reconstitution of EGFR ubiquitination implies that the RING finger plays an essential direct role in ubiquitin ligation. Our results attribute to the RING finger of c-Cbl a causative role in endocytic sorting of EGFR and desensitization of signal transduction.     

Conjugation to Nedd8 instigates ubiquitylation and down-regulation of activated receptor tyrosine kinases

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.     

Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.     

Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain

Ligand-induced down-regulation of the epidermal growth factor receptor (EGFR) comprises activation of two sequential transport steps. The first involves endocytic uptake by clathrin-coated vesicles, the second transfer of endocytosed EGFR from endosomes to lysosomes. Here we demonstrate that the second transport step requires a domain of the EGFR that encompasses residues 985-996 and was previously found to interact with actin. Deletion of domain 989-994 (Delta989-994 EGFR) did not interfere with EGFR uptake but completely abrogated its degradation. In contrast, both uptake and degradation were affected for K721A EGFR, a kinase-deficient EGFR mutant. To measure intracellular EGFR sorting, we developed a novel cell fractionation assay toward which cells were co-transfected for chicken hepatic lectin, a receptor for agialoglycoproteins. These cells were incubated with agialofetuin-coupled colloidal gold, which was targeted to lysosomes after receptor-mediated endocytosis. Compartments within the lysosomal pathway gained buoyant density because of the presence of colloidal gold and could be isolated from cell homogenates by ultracentrifugation through a high-density sucrose cushion. In contrast to endocytosed wild type EGFR, both Delta989-994 EGFR and K721A EGFR were largely not retrieved in gold-containing endocytic compartments. These results are supported with morphological data. We conclude that sorting of endocytosed EGFR into the degradation pathway requires both its kinase activity and actin-binding domain.     

Role of cyclic GMP and calcineurin in homologous and heterologous desensitization of natriuretic peptide receptor-A

The natriuretic peptide receptor-A (NPR-A) mediates natriuretic, hypotensive, and antihypertrophic effects of natriuretic peptides through the production of cGMP. In pathological conditions such as heart failure, these effects are attenuated by homologous and heterologous desensitization mechanisms resulting in the dephosphorylation of the cytosolic portion of the receptor. In contrast with natriuretic peptide-induced desensitization, pressor hormone-induced desensitization is dependent on protein kinase C (PKC) stimulation and (or) cytosolic calcium elevation. Mechanisms by which PKC and Ca(2+) promote NPR-A desensitization are not known. The role of cGMP and of the cytosolic Ca(2+) pathways in NPR-A desensitization were therefore studied. In contrast with the activation of NPR-A by its agonist, activation of soluble guanylyl cyclases of LLC-PK1 cells by sodium nitroprusside also leads to a production of cGMP but without altering NPR-A activation. Consequently, cGMP elevation per se does not appear to mediate homologous desensitization of NPR-A. In addition, cytosolic calcium increase is required only for the heterologous desensitization pathway since the calcium chelator BAPTA-AM blocks only PMA or ionomycin-induced desensitization. Calcineurin inhibitors block the NPR-A guanylyl cyclase heterologous desensitization induced by ionomycin, suggesting an essential role for this Ca(2+)-stimulated phosphatase in NPR-A desensitization. In summary, the present report demonstrates that neither cGMP nor Ca(2+) cytosolic elevation cause NPR-A homologous desensitization. Our results also indicate for the first time a role for calcineurin in NPR-A heterologous desensitization.     

The relationship between ER-multivesicular body membrane contacts and the ESCRT machinery

Activated EGFR (epidermal growth factor receptor) undergoes ESCRT (endosomal sorting complex required for transport)-mediated sorting on to ILVs (intraluminal vesicles) of endosomes before degradation in the lysosome. Sorting of endocytosed EGFR on to ILVs removes the catalytic domain of the EGFR from the cytoplasm, resulting in termination of receptor signalling. EGFR signalling is also subject to down-regulation through receptor dephosphorylation by the ER (endoplasmic reticulum)-localized PTP1B (protein tyrosine phosphatase 1B). PTP1B on the cytoplasmic face of the ER interacts with endocytosed EGFR via direct membrane contacts sites between the ER and endosomes. In the present paper, we review the relationship between ER-endosome membrane contact sites and ILV formation, and their potential role in the regulation of EGFR sorting on to ILVs, through PTP1B-mediated dephosphorylation of both EGFR and components of the ESCRT machinery.     

Identification of two distinct pathways of protein kinase Calpha down-regulation in intestinal epithelial cells

Signal transduction pathways are controlled by desensitization mechanisms, which can affect receptors and/or downstream signal transducers. It has long been recognized that members of the protein kinase C (PKC) family of signal transduction molecules undergo down-regulation in response to activation. Previous reports have indicated that key steps in PKCalpha desensitization include caveolar internalization, priming site dephosphorylation, ubiquitination of the dephosphorylated protein, and degradation by the proteasome. In the current study, comparative analysis of PKCalpha processing induced by the PKC agonists phorbol 12-myristate 13-acetate and bryostatin 1 in IEC-18 rat intestinal epithelial cells demonstrates that: (a) at least two pathways of PKCalpha down-regulation can co-exist within cells, and (b) a single PKC agonist can activate both pathways at the same time. Using a combined biochemical and morphological approach, we identify a novel pathway of PKCalpha desensitization that involves ubiquitination of mature, fully phosphorylated activated enzyme at the plasma membrane and subsequent down-regulation by the proteasome. The phosphatase inhibitors okadaic acid and calyculin A accelerated PKCalpha down-regulation and inhibitors of vesicular trafficking did not prevent degradation of the protein, indicating that neither internalization nor priming site dephosphorylation are requisite intermediate steps in this ubiquitin/proteasome dependent pathway of PKCalpha down-regulation. Instead, caveolar trafficking and dephosphorylation are involved in a second, proteasome-independent mechanism of PKCalpha desensitization in this system. Our findings highlight subcellular distribution and phosphorylation state as critical determinants of PKCalpha desensitization pathways.     

Ca2+-independent protein kinase Cs mediate heterologous desensitization of leukocyte chemokine receptors by opioid receptors

Heterologous desensitization of chemokine receptors by opioids has been considered to contribute to their immunosuppressive effects. Previous studies show that Met-enkephalin, an endogenous opioid, down-regulates chemotaxis of selected chemokine receptors via phosphorylation. In the present study, we further investigated the molecular mechanism of such cross-regulation. Our data showed that preincubation with Met-enkephalin inhibited both MIP-1 alpha-mediated chemotaxis and Ca(2+) flux of monocytes in a dose-dependent manner. The inhibitory effects were maximal using nanomolar concentrations of activating chemokines, a concentration found in physiological conditions. A decrease both in chemokine receptor affinity and in coupling efficiency between receptors and G protein were observed, which directly contributed to the desensitization effects. However, comparing with chemokines such as MIP-1 alpha and MCP-1, opioids did not elicit a calcium flux, failed to induce MIP-1 alpha receptors internalization, and mediated a less potent heterologous desensitization. We hypothesized that these differences might originate from the involvement of different protein kinase C (PKC) isotypes. In our studies, opioid-mediated down-regulation of MIP-1 alpha receptors could be blocked by the general PKC inhibitor calphostin C, but not by the calcium-dependent classic PKC inhibitor Go6976. Western blotting analysis and immunofluorescent staining further showed that only calcium-independent PKCs were activated upon opioid stimulation. Thus, opioids achieve desensitization of chemokine receptors via a unique pathway, involving only calcium-independent PKC isotypes.     

Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of (125)I-EGF internalization, whereas internalization of (125)I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized (125)I-EGF was increased, while degradation of (125)I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.     

A mutant EGF-receptor defective in ubiquitylation and endocytosis unveils a role for Grb2 in negative signaling

Ligand-induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c-Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c-Cbl, here we report that defective Tyr1045 of EGFR, an inducible c-Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand-induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization-defective mutant receptor imply that c-Cbl-mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displayed significant residual ligand-induced ubiquitylation, especially in the presence of an overexpressed c-Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c-Cbl complex to Grb2-specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well-characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c-Cbl-dependent sorting of active tyrosine kinases to destruction.     

Adenovirus RIDbeta subunit contains a tyrosine residue that is critical for RID-mediated receptor internalization and inhibition of Fas- and TRAIL-induced apoptosis

The adenovirus-encoded receptor internalization and degradation (RID) protein (previously named E3-10.4K/14.5K), which is composed of RIDalpha and RIDbeta subunits, down-regulates a number of cell surface receptors in the tumor necrosis factor (TNF) receptor superfamily, namely Fas, TRAIL receptor 1, and TRAIL receptor 2. Down-regulation of these "death" receptors protects adenovirus-infected cells from apoptosis induced by the death receptor ligands Fas ligand and TRAIL. RID also down-regulates certain tyrosine kinase cell surface receptors, especially the epidermal growth factor receptor (EGFR). RID-mediated Fas and EGFR down-regulation occurs via endocytosis of the receptors into endosomes followed by transport to and degradation within lysosomes. However, the molecular interactions underlying this function of RID are unknown. To investigate the molecular determinants of RIDbeta that are involved in receptor down-regulation, mutations within the cytoplasmic tail of RIDbeta were constructed and the mutant proteins were analyzed for their capacity to internalize and degrade Fas and EGFR and to protect cells from death receptor ligand-induced apoptosis. The results demonstrated the critical nature of a tyrosine residue near the RIDbeta C terminus; mutation of this residue to alanine abolished RID function. Mutating the tyrosine to phenylalanine did not abolish the function of RID, arguing that phosphorylation of the tyrosine is not required for function. These data suggest that this tyrosine residue forms part of a tyrosine-based sorting signal (Yxxphi). Additional mutations that target another potential sorting motif and several possible protein-protein interaction motifs had no discernible effect on RID function. It was also demonstrated that mutation of serine 116 to alanine eliminated phosphorylation of RIDbeta but did not affect any of the functions of RID that were examined. These results suggest a model in which the tyrosine-based sorting signal in RID plays a role in RID's ability to down-regulate receptors.     

Agonist-dependent μ-opioid receptor signaling can lead to heterologous desensitization

Desensitization of the µ-opioid receptor (MOR) has been implicated as an important regulatory process in the development of tolerance to opiates. Monitoring the release of intracellular Ca²⁺ ([Ca²⁺]_i), we reported that [D-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin (DAMGO)-induced receptor desensitization requires receptor phosphorylation and recruitment of β-arrestins (βArrs), while morphine-induced receptor desensitization does not. In current studies, we established that morphine-induced MOR desensitization is protein kinase C (PKC)-dependent. By using RNA interference techniques and subtype specific inhibitors, PKCε was shown to be the PKC subtype activated by morphine and the subtype responsible for morphine-induced desensitization. In contrast, DAMGO did not increase PKCε activity and DAMGO-induced MOR desensitization was not affected by modulating PKCε activity. Among the various proteins within the receptor signaling complex, Gαi2 was phosphorylated by morphine-activated PKCε. Moreover, mutating three putative PKC phosphorylation sites, Ser⁴⁴, Ser¹⁴⁴ and Ser³⁰² on Gαi2 to Ala attenuated morphine-induced, but not DAMGO-induced desensitization. In addition, pretreatment with morphine desensitized cannabinoid receptor CB1 agonist WIN 55212-2-induced [Ca²⁺]_i release, and this desensitization could be reversed by pretreating the cells with PKCε inhibitor or overexpressing Gαi2 with the putative PKC phosphorylation sites mutated. Thus, depending on the agonist, activation of MOR could lead to heterologous desensitization and probable crosstalk between MOR and other Gαi-coupled receptors, such as the CB1.     

Neurotensin triggers dopamine D2 receptor desensitization through a protein kinase C and beta-arrestin1-dependent mechanism

The peptide neurotensin (NT) is known to exert a potent excitatory effect on the dopaminergic system by inhibiting D2 dopamine (DA) receptor (D2R) function. This regulation is dependent on activation of PKC, a well known effector of the type 1 NT receptor (NTR1). Because PKC phosphorylation of the D2R has recently been shown to induce its internalization, we hypothesized that NT acts to reduce D2R function through heterologous desensitization of the D2R. In the present study, we first used HEK-293 cells to demonstrate that NT induces PKC-dependent D2R internalization. Furthermore, internalization displayed faster kinetics in cells expressing the D2R short isoform, known to act as an autoreceptor in DA neurons, than in cells expressing the long isoform, known to act as a postsynaptic D2R. In patch clamp experiments on cultured DA neurons, overexpression of a mutant D2S lacking three key PKC phosphorylation sites abrogated the ability of NT to reduce D2R-mediated cell firing inhibition. Short interfering RNA-mediated inhibition of β-arrestin1 and dynamin2, proteins important for receptor desensitization, reduced agonist-induced desensitization of D2R function, but only the inhibition of β-arrestin1 reduced the effect of NT on D2R function. Taken together, our data suggest that NT acutely regulates D2 autoreceptor function and DA neuron excitability through PKC-mediated phosphorylation of the D2R, leading to heterologous receptor desensitization.     

Endocytosis and intracellular trafficking of ErbBs

This review article describes the pathways and mechanisms of endocytosis and post-endocytic sorting of the EGF receptor (EGFR/ErbB1) and other members of the ErbB family. Growth factor binding to EGFR accelerates its internalization through clathrin-coated pits which is followed by the efficient lysosomal targeting of internalized receptors and results in receptor down-regulation. The role of EGFR interaction with the Grb2 adaptor protein and Cbl ubiquitin ligase, and receptor ubiquitination in the clathrin-dependent internalization and sorting of EGFR in multivesicular endosomes is discussed. Activation and phosphorylation of ErbB2, ErbB3 and ErbB4 also results in their ubiquitination. However, these ErbBs are internalized and targeted to lysosomes less efficiently than EGFR. When overexpressed endocytosis-impaired ErbBs may inhibit the internalization and degradation of EGFR.     

Homologous and heterologous desensitization of guanylyl cyclase-B signaling in GH3 somatolactotropes

The guanylyl cyclases, GC-A and GC-B, are selective receptors for atrial and C-type natriuretic peptides (ANP and CNP, respectively). In the anterior pituitary, CNP and GC-B are major regulators of cGMP production in gonadotropes and yet mouse models of disrupted CNP and GC-B indicate a potential role in growth hormone secretion. In the current study, we investigate the molecular and pharmacological properties of the CNP/GC-B system in somatotrope lineage cells. Primary rat pituitary and GH3 somatolactotropes expressed functional GC-A and GC-B receptors that had similar EC₅₀ properties in terms of cGMP production. Interestingly, GC-B signaling underwent rapid homologous desensitization in a protein phosphatase 2A (PP2A)-dependent manner. Chronic exposure to either CNP or ANP caused a significant down-regulation of both GC-A- and GC-B-dependent cGMP accumulation in a ligand-specific manner. However, this down-regulation was not accompanied by alterations in the sub-cellular localization of these receptors. Heterologous desensitization of GC-B signaling occurred in GH3 cells following exposure to either sphingosine-1-phosphate or thyrotrophin-releasing hormone (TRH). This heterologous desensitization was protein kinase C (PKC)-dependent, as pre-treatment with GF109203X prevented the effect of TRH on CNP/GC-B signaling. Collectively, these data indicate common and distinct properties of particulate guanylyl cyclase receptors in somatotropes and reveal that independent mechanisms of homologous and heterologous desensitization occur involving either PP2A or PKC. Guanylyl cyclase receptors thus represent potential novel therapeutic targets for treating growth-hormone-associated disorders.     

Endocytosis and intracellular trafficking of ErbBs

This review article describes the pathways and mechanisms of endocytosis and post-endocytic sorting of the EGF receptor (EGFR/ErbB1) and other members of the ErbB family. Growth factor binding to EGFR accelerates its internalization through clathrin-coated pits which is followed by the efficient lysosomal targeting of internalized receptors and results in receptor down-regulation. The role of EGFR interaction with the Grb2 adaptor protein and Cbl ubiquitin ligase, and receptor ubiquitination in the clathrin-dependent internalization and sorting of EGFR in multivesicular endosomes is discussed. Activation and phosphorylation of ErbB2, ErbB3 and ErbB4 also results in their ubiquitination. However, these ErbBs are internalized and targeted to lysosomes less efficiently than EGFR. When overexpressed endocytosis-impaired ErbBs may inhibit the internalization and degradation of EGFR.     

Basolateral EGF Receptor Sorting Regulated by Functionally Distinct Mechanisms in Renal Epithelial Cells

Proliferation of epithelial tissues is controlled by polarized distribution of signaling receptors including the EGF receptor (EGFR). In kidney, EGFRs are segregated from soluble ligands present in apical fluid of nephrons by selective targeting to basolateral membranes. We have shown previously that the epithelial‐specific clathrin adaptor AP1B mediates basolateral EGFR sorting in established epithelia. Here we show that protein kinase C (PKC)‐dependent phosphorylation of Thr654 regulates EGFR polarity as epithelial cells form new cell–cell junctional complexes. The AP1B‐dependent pathway does not override a PKC‐resistant T654A mutation, and conversely AP1B‐defective EGFRs sort basolaterally by a PKC‐dependent mechanism, in polarizing cells. Surprisingly, EGFR mutations that interfere with these different sorting pathways also produce very distinct phenotypes in three‐dimensional organotypic cultures. Thus EGFRs execute different functions depending on the basolateral sorting route. Many renal disorders have defects in cell polarity and the notion that apically mislocalized EGFRs promote proliferation is still an attractive model to explain many aspects of polycystic kidney disease. Our data suggest EGFR also integrates various aspects of polarity by switching between different basolateral sorting programs in developing epithelial cells. Fundamental knowledge of basic mechanisms governing EGFR sorting therefore provides new insights into pathogenesis and advances drug discovery for these renal disorders.     

Role of specific protein kinase C isoforms in modulation of beta1- and beta2-adrenergic receptors

The function of beta-adrenergic receptor (betaAR) is modulated by the activity status of alpha1-adrenergic receptors (alpha1ARs) via molecular crosstalk, and this becomes evident when measuring cardiac contractile responses to adrenergic stimulation. The molecular mechanism underlying this crosstalk is unknown. We have previously demonstrated that overexpression of alpha1B-adrenergic receptor (alpha1BAR) in transgenic mice leads to a marked desensitization of betaAR-mediated adenylyl cyclase stimulation which is correlated with increased levels of activated protein kinase C (PKC) beta, delta and [J. Mol. Cell. Cardiol. 30 (1998) 1827]. Therefore, we wished to determine which PKC isoforms play a role in heterologous betaAR desensitization and also which isoforms of the betaAR were the molecular target(s) for PKC. In experiments using constitutively activated PKC expression constructs transfected into HEK 293 cells also expressing the beta2AR, constitutively active (CA)-PKC overexpression was first confirmed by immunoblots using specific anti-PKC antibodies. We then demonstrated that the different PKC subtypes lead to a decreased maximal cAMP accumulation following isoproterenol stimulation with a rank order of PKCalpha > or = PKCzeta>PKC>PKCbetaII. However, a much more dramatic desensitization of adenylyl cyclase stimulation was observed in cells co-transfected with different PKC isoforms and beta1AR. Further, the modulation of beta1AR by PKC isoforms had a different rank order than for the beta2AR: PKCbetaII>PKCalpha>PKC>PKCzeta. PKC-mediated desensitization was reduced by mutating consensus cAMP-dependent protein kinase (PKA)/PKC sites in the third intracellular loop and/or the carboxy-terminal tail of either receptor. Our results demonstrate therefore that the beta1AR is the most likely molecular target for PKC-mediated heterologous desensitization in the mammalian heart and that modulation of adrenergic receptor activity in any given cell type will depend on the complement of PKC isoforms present.