The T-cell antigen CD5 acts as a receptor and substrate for the protein-tyrosine kinase p56lck.

CD5 is a T-cell-specific antigen which binds to the B-cell antigen CD72 and acts as a coreceptor in the stimulation of T-cell growth. CD5 associates with the T-cell receptor zeta chain (TcR zeta)/CD3 complex and is rapidly phosphosphorylated on tyrosine residues as a result of TcR zeta/CD3 ligation. However, despite this, the mechanism by which CD5 generates intracellular signals is unclear. In this study, we demonstrate that CD5 is coupled to the protein-tyrosine kinase p56lck and can act as a substrate for p56lck. Coexpression of CD5 with p56lck in the baculovirus expression system resulted in the phosphorylation of CD5 on tyrosine residues. Further, anti-CD5 and anti-p56lck coprecipitated each other in a variety of detergents, including Nonidet P-40 and Triton X-100. Anti-CD5 also precipitated the kinase from various T cells irrespective of the expression of TcR zeta/CD3 or CD4. No binding between p59fyn(T) and CD5 was detected in T cells. The binding of p56lck to CD5 induced a 10- to 15-fold increase in p56lck catalytic activity, as measured by in vitro kinase analysis. In vivo labelling with 32P(i) also showed a four- to fivefold increase in Y-394 occupancy in p56lck when associated with CD5. The use of glutathione S-transferase-Lck fusion proteins in precipitation analysis showed that the SH2 domain of p56lck could recognize CD5 as expressed in the baculovirus expression system. CD5 interaction with p56lck represents a novel variant of a receptor-kinase complex in which receptor can also serve as substrate. The CD5-p56lck interaction is likely to play roles in T-cell signalling and T-B collaboration.     

Coassociation of CD26 (dipeptidyl peptidase IV) with CD45 on the surface of human T lymphocytes.

In the present report, we demonstrated that modulation of CD26 from T cell surface induced by antiCD26 (1F7) led to enhanced phosphorylation of CD3 zeta tyrosine residues and increased CD4 associated p56lck tyrosine kinase activity. We further showed that CD26 was comodulated on the T cell surface with CD45, a known membrane-linked protein tyrosine phosphatase and that anti-CD26 was capable of precipitating CD45 from T cell lysates. These findings strongly suggest that CD26 may be closely associated with the CD45 protein tyrosine phosphatase on T cell surface and further support the notion that the interaction of CD26 with CD45 results in enhanced tyrosine kinase activity, zeta chain phosphorylation, and T cell activation.     

The tyrosine kinase lck is critically involved in the growth transformation of human B lymphocytes.

Epstein-Barr virus (EBV) exposure of human B lymphocytes induces rapid, Ca(2+)-dependent tyrosine phosphorylation of two cytosolic proteins, one likely the CD21 EBV receptor and another unknown species of 55-60 kDa. We now identify the latter protein as the tyrosine kinase lck (p56lck). In T cells many activation events reduce the high constitutive p56lck expression levels typical for that lineage, and they induce the appearance of a 60-kDa lck species. We now demonstrate that in B cells exposed to EBV the at best low constitutive p56lck expression levels are rapidly and transiently up-regulated without generation of 60-kDa lck. lck-specific antisense oligonucleotides block p56lck induction and prevent subsequent B cell activation and immortalization whereas B cell activation by nononcogenic agents was unaffected. We propose that p56lck superinduction is a transformation prerequisite which signals entry into the oncogenic growth transformation process.     

Biochemical analysis of interleukin-2 receptor beta chain phosphorylation by p56(lck)

Tyrosine phosphorylation of multiple proteins, including the receptor itself, is an initial event in IL-2 signaling and leads to recruitment of SH2 or PTB domain-containing proteins to the receptor. In this study, we have used subdomains of the IL-2 receptor beta chain (IL-2Rbeta) expressed in Escherichia coli as GST fusion proteins to identify the tyrosine residues that could be phosphorylated by p56(lck), one of the critical tyrosine kinases activated by IL-2. We report that recombinant p56(lck) phosphorylates in vitro tyrosine residues within the IL-2Rbeta chain but not those within the IL-2Rgamma chain. p56(lck) phosphorylates tyrosine residues 355, 358 and 361 but not 338 of the IL-2Rbeta chain acidic subdomain. Interestingly, phosphorylation of Tyr-358 appears to require the presence of either Tyr-355 or Tyr-361. p56(lck) also phosphorylates very efficiently the two tyrosines present in the IL-2Rbeta chain C-terminal region, Tyr-392 and Tyr-510. We also investigated the association of p56(lck) with the IL-2Rbeta chain which was found to depend on a short stretch of the IL-2Rbeta chain acidic subdomain, and to be independent of the presence of its tyrosine residues.     

Inhibition of p56(lck) tyrosine kinase by isothiazolones

Lck encodes a 56-kDa protein-tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56(lck), an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56(lck) kinase activity with IC50 = 1-7 microM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-Jun N-terminal kinase 2alpha. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50 = 35 microM) and blocked T cell proliferation in response to alloantigen (IC50 = 14 microM) and CD3/CD28-induced IL-2 secretion (IC50 = 2.2 microM) in primary T cell cultures. Inhibition of p56(lck )by A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56(lck) kinase activity and TCR-dependent signal transduction. Incubation with thiols such as beta-ME or DTT also blocked the ability of the isothiazolone to inhibit p56(lck) kinase activity. LC/MS analysis established the covalent modification of p56(lck) at cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56(lck) catalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56(lck) enzyme. Loss of p56(lck) activity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56(lck) inhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.     

A lymphocyte-specific protein tyrosine kinase, p56lck, regulates the PMA-induced internalization of CD4.

p56lck, a member of the src family of non-receptor protein tyrosine kinases (PTKs), is expressed predominantly in T-lymphocytes. Association of p56lck with CD4 and CD8 T-cell receptor (TcR) accessory molecules suggests that p56lck may play a specialized role in antigen-induced T-cell activation. CD4 and CD8 molecules are known to stabilize the interaction between TcR and the major histocompatibility complex during T-cell activation. To examine the role of p56lck in the dynamics of the CD4 molecule, p56lck-expressing transfectant cell clones were prepared by the transfection of an lck-gene plasmid containing an inducible promoter into a CD4+lck- human monocytoid cell line. When these transfectant cells were stimulated with phorbol ester, CD4 internalization on these p56lck-expressing cell lines was selectively and markedly retarded, as compared to p56lck-negative control cell lines. When cell-surface CD4 and intracellular CD4 were selectively precipitated after stimulation, the intracellular CD4 molecules were dissociated from p56lck whereas the surface-retained CD4 molecules were still associated with p56lck. Moreover, the dissociation of p56lck from CD4 appeared to occur prior to the PMA-induced internalization of CD4. These data indicate that p56lck regulates the PMA-induced internalization of CD4 possibly via its association with CD4. Treatment with genistein, a PTK inhibitor, revealed that the PTK activity of p56lck might not be involved in this regulatory effect of p56lck on CD4 internalization.     

Inhibition of T-cell receptor beta-chain gene rearrangement by overexpression of the non-receptor protein tyrosine kinase p56lck.

The variable region genes of the T cell receptor (TCR) alpha and beta chains are assembled by somatic recombination of separate germline elements. During thymocyte development, gene rearrangements display both an ordered progression, with beta chain formation preceding alpha chain, and allelic exclusion, with each cell containing a single functional beta chain rearrangement. Although considerable evidence supports the view that the individual loci are regulated independently, signaling molecules that may participate in controlling TCR gene recombination remain unidentified. Here we report that the lymphocyte-specific protein tyrosine kinase p56lck, when overexpressed in developing thymocytes, provokes a reduction in V beta--D beta rearrangement while permitting normal juxtaposition of other TCR gene segments. Our data support a model in which p56lck activity impinges upon a signaling process that ordinarily permits allelic exclusion at the beta-chain locus.     

Differential association of CD45 isoforms with CD4 and CD8 regulates the actions of specific pools of p56lck tyrosine kinase in T cell antigen receptor signal transduction.

An investigation into the role of CD45 isoforms in T cell antigen receptor signal transduction was carried out by transfecting CD45-negative CD4(+)CD8(+) HPB-ALL T cells with the CD45R0, CD45RBC, and CD45RABC isoforms. Fluorescence resonance energy transfer analysis showed that the CD45R0 isoform, but not the CD45RBC or CD45RABC isoforms, was found as homodimers and also preferentially associated with CD4 and CD8 at the cell-surface. A comparison was therefore made of T cell antigen receptor signaling between sub-clones expressing either CD45R0 or CD45RBC. Under basal conditions CD4-associated p56(lck) tyrosine kinase activity and cellular protein tyrosine phosphorylation levels were higher in the CD45R0(+) than in the CD45RBC(+) sub-clones. Upon CD3-CD4 ligation, TCR-zeta phosphorylation, ZAP-70 recruitment to the p21/p23 TCR-zeta phosphoisomers, ZAP-70 phosphorylation, as well as p56(lck), c-Cbl and Slp-76 phosphorylation, were all markedly increased in CD45R0(+) compared with CD45RBC(+) cells. T cell antigen receptor (TCR) stimulation alone also promoted c-Cbl phosphorylation in CD45R0(+) but not in CD45RBC(+) cells. Our results are consistent with a model in which association of CD45R0 with CD4 generates a more active pool of CD4-associated p56(lck) kinase molecules. Upon CD3-CD4 co-ligation, the active p56(lck) increases the intensity of T cell antigen receptor signal transduction coupling by promoting TCR-zeta chain phosphorylation and ZAP-70 recruitment.     

The protein tyrosine kinase p56lck inhibits CD4 endocytosis by preventing entry of CD4 into coated pits

The lymphocyte glycoprotein CD4 is constitutively internalized and recycled in nonlymphoid cells, but is excluded from the endocytic pathway in lymphocytic cells (Pelchen-Matthews, A., J. E. Armes, G. Griffiths, and M. Marsh. 1991. J. Exp. Med. 173: 575-587). Inhibition of CD4 endocytosis is dependent on CD4 expressing an intact cytoplasmic domain and is only observed in cells where CD4 can interact with the protein tyrosine kinase p56lck, a member of the src gene family. We have expressed p56lck, p60c-src, or chimeras of the two proteins in CD4-transfected NIH-3T3 or HeLa cells. Immunoprecipitation of CD4 and in vitro kinase assays showed that p56lck and the lck/src chimera, which contains the NH2 terminus of p56lck, can associate with CD4. In contrast, p60c-src and the src/lck chimera, which has the NH2 terminus of p60c-src, do not associate with CD4. Endocytosis assays using radioiodinated anti-CD4 monoclonal antibodies demonstrated that coexpression of CD4 with p56lck, but not with p60c-src, inhibited CD4 endocytosis, and that the extent of the inhibition depended directly on the relative levels of CD4 and p56lck expressed. The uptake of mutant CD4 molecules which cannot interact with p56lck was not affected. Measurement of the fluid-phase endocytosis of HRP or the internalization of transferrin indicated that the effect of p56lck was specific for CD4, and did not extend to other receptor-mediated or fluid-phase endocytic processes. Immunogold labeling of CD4 at the cell surface and observation by electron microscopy demonstrated directly that p56lck inhibits CD4 endocytosis by preventing its entry into coated pits.     

Activated p56lck directs maturation of both CD4 and CD8 single-positive thymocytes

p56(lck) is a protein tyrosine kinase expressed throughout T cell development. It associates noncovalently with the cytoplasmic domains of the CD4 and CD8 coreceptor molecules and has been implicated in TCR signaling in mature T cells. Its role in early thymocyte differentiation has been demonstrated in vivo, both by targeted gene disruption and by transgene expression. Previously, we showed that expression of a dominant-negative form of p56(lck) in double-positive thymocytes inhibits positive selection. We now demonstrate that expression of constitutively activated p56(lck) (p56(lck)F505) accelerates the transition from the double-positive to the single-positive stage. Importantly, p56(lck)F505 drives survival and lineage commitment of thymocytes in the absence of TCR engagement by appropriate MHC molecules. These results indicate that activation of p56(lck) constitutes an early step in conveying maturational signals after TCR ligation by a positively selecting ligand. Our study provides direct in vivo evidence for the role of p56(lck) in regulating TCR signaling.     

Essential role of CD8 palmitoylation in CD8 coreceptor function

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.     

The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck

The CD4 and CD8 T cell antigens are thought to transduce an independent signal during the process of T cell activation. We report our evaluation of the possible involvement of the lymphocyte-specific tyrosine kinase p56lck in these transduction pathways. Our data demonstrate that p56lck is specifically modulated with either CD4 or CD8 following antibody-mediated cross-linking of these molecules and that a large fraction of the total cellular lck protein can be coimmunoprecipitated with these surface glycoproteins. These results suggest that p56lck is functionally and physically associated with CD4/CD8 in normal murine T lymphocytes and support the concept that an independent signal is transduced by the interaction of these surface molecules with major histocompatibility complex determinants.     

Effect of human immunodeficiency virus gp120 glycoprotein on the association of the protein tyrosine kinase p56lck with CD4 in human T lymphocytes

The human immunodeficiency virus binds to CD4+ T lymphocytes through the interaction of its envelope glycoprotein (gp120) with the CD4 molecule. The src-related protein tyrosine kinase p56lck is physically associated with CD4 and is co-immunoprecipitated by CD4 monoclonal antibody (mAb). Activators of protein kinase C (PKC) cause the dissociation of p56lck from CD4. Here we report that gp120 mAb immunoprecipitated the p56lck.CD4.gp120 complex after short term treatment (20 min) of human T lymphocytes with gp120. The p56lck that was associated with the CD4.gp120 complex was dissociated by activators of PKC. This effect was abolished by pretreatment of cells with PKC inhibitors. Thus the p56lck.CD4.gp120 immune complex immunoprecipitated by gp120 mAb behaves in a similar manner, with respect to PKC activation or inhibition, to the p56lck.CD4 complex immunoprecipitated by CD4 mAb. Short term treatment of cells with gp120, followed by gp120 mAb, resulted in an increase in the tyrosine kinase activity of p56lck associated with CD4. However, the amount of enzyme associated with CD4 remained unchanged. Long term treatment (20 h) of human T lymphocytes with gp120 resulted in the down-regulation of cell surface CD4 molecules. A parallel decrease in CD4-associated gp120 was also observed. In addition, gp120 caused the dissociation of p56lck and CD4. However, the dissociation of the p56lck from CD4 occurred at much faster rate than the down-regulation of surface CD4 molecules. Such mechanisms may account for the down-regulation of cell surface CD4 molecules and the depletion of functional CD4+ T lymphocytes which are characteristic of human immunodeficiency virus infections and acquired immune deficiency syndrome pathogenesis.     

Activated lck tyrosine protein kinase stimulates antigen-independent interleukin-2 production in T cells.

p56lck, a member of the src family of cytoplasmic tyrosine kinases, is expressed predominantly in T cells where it associates with the T-cell surface molecules CD4 and CD8. Mutants of CD4 and CD8 that have lost the ability to associate with p56lck no longer enhance antigen-induced T-cell activation. This suggests that p56lck plays an important role during T-cell activation. In an effort to understand the function of p56lck in T cells, a constitutively activated lck gene (F505lck) was introduced into T-helper hybridoma cell lines by retroviral infection. In four T-cell lines we examined, the activated lck protein stimulated interleukin-2 (IL-2) production, a hallmark of T-cell activation, in the absence of antigenic stimulation. In addition, a marked increase in antigen-independent IL-2 production was apparent when T cells infected with a temperature-sensitive F505lck were shifted to the permissive temperature. Only one cell line expressing F505lck exhibited increased sensitivity to antigenic stimulation. The SH3 domain of p56lck was dispensable for the induction of antigen-independent IL-2 production. In contrast, deletion of the majority of the SH2 domain of p56F505lck reduced its ability to induce spontaneous IL-2 production markedly. Activated p60c-src also induced antigen-independent IL-2 production, whereas two other tyrosine kinases, v-abl and the platelet-derived growth factor receptor, did not. Tyrosine phosphorylation of a 70-kDa cellular protein was observed after cross-linking of CD4 in T cells expressing F505lck but not in cells expressing F527src.     

p56lck stably associates with a 115 kDa substrate.

Using antibodies directed against p56lck, we have identified a 115 kDa protein (p115) that is specifically immunoprecipitated with p56lck from whole cell lysates. The p56lck/p115 complex is stable in the presence of nonionic detergents. p115 becomes phosphorylated on tyrosine residues in p56lck immune-complex kinase assays. Treatment of whole cells with 12-O-tetradecanoyl phorbol-13-acetate decreases the subsequent tyrosine phosphorylation of p115 in immune-complex kinase assays.     

Myxoma virus induces extensive CD4 downregulation and dissociation of p56lck in infected rabbit CD4+ T lymphocytes.

Myxoma virus is a pathogenic poxvirus that induces extensive dysregulation of cellular immunity in infected European rabbits. Infection of a rabbit CD4+ T-cell line (RL-5) with myxoma virus results in dramatic reductions of cell surface levels of CD4 as monitored by flow cytometry. The virus-induced downregulation of CD4 requires early but not late viral gene expression and could not be inhibited by staurosporine, an inhibitor of protein kinase C, which effectively blocks phorbol 12-myristate-13-acetate-induced downregulation of CD4. The decrease in total cellular levels of CD4 during myxoma virus infection could be inhibited by the lysosomotrophic agent NH4Cl, suggesting a lysosomal fate for CD4 during myxoma virus infection. Steady-state levels of the CD4-associated protein tyrosine kinase p56lck remained unchanged during myxoma virus infection, suggesting that p56lck dissociates from CD4 prior to CD4 degradation in virus infected cells. Total p56lck kinase activity was unaffected during myxoma virus infection, although the amount of p56lck physically associated with CD4 declined in parallel with the loss of CD4. Thus, myxoma virus infection of CD4+ T lymphocytes triggers CD4 downregulation via a protein kinase C-independent pathway, causing the dissociation of p56lck and the degradation of CD4 in lysosomal vesicles.     

Oncogenic activation of the Lck protein accompanies translocation of the LCK gene in the human HSB2 T-cell leukemia.

The tyrosine protein kinase p56lck transduces signals important for antigen-induced T-cell activation. In transgenic mice, p56lck is oncogenic when overexpressed or expressed as a mutant, catalytically activated enzyme. In humans, the LCK gene is located at the breakpoint of the t(1;7)(p34;q34) chromosomal translocation. This translocation positions the beta T-cell receptor constant region enhancer upstream of the LCK gene without interrupting the LCK coding sequences, and a translocation of this sort occurs in both the HSB2 and the SUP-T-12 T-cell lines. We have found that, although the level of the p56lck protein in HSB2 cells is elevated approximately 2-fold in comparison with that in normal T-cell lines, total cellular tyrosine protein phosphorylation is elevated approximately 10-fold. Increased levels of phosphotyrosine in HSB2 cells resulted from mutations in the LCK gene that activated its function as a phosphotransferase and converted it into a dominant transforming oncogene. The oncogenic p56lck in HSB2 cells contained one amino acid substitution within the CD4/CD8-binding domain, two substitutions in the kinase domain, and an insertion of Gln-Lys-Pro (QKP) between the SH2 and kinase domains. In NIH 3T3 fibroblasts, three of these mutations cooperated to produce the fully oncogenic form of this p56lck variant. These results suggest that mutation of LCK may contribute to some human T-cell leukemias.     

Purification and initial characterization of the lymphocyte-specific protein-tyrosyl kinase p56lck from a baculovirus expression system.

A baculovirus expression system has been used to express large quantities of the lymphocyte-specific protein-tyrosyl kinase p56lck. A series of chromatographic steps, including the novel application of metalchelate affinity chromatography for protein kinase purification, were employed to obtain p56lck in a highly active form. Recombinant p56lck was purified to apparent homogeneity as determined by polyacrylamide gel electrophoretic analyses and was found to migrate in SDS gels as two related species, both with apparent molecular masses close to 56 kDa. p56lck phosphorylated all assayed substrates exclusively on tyrosyl residues, and underwent autophosphorylation at one principal site, also on a tyrosyl residue. p56lck displayed a high affinity for a synthetic peptide corresponding to the cytoplasmic domain (residues 52-164) of the T-cell receptor zeta-chain (TCR-zeta) (Km approximately 6.5 microM) but a low affinity for a peptide corresponding to its own autophosphorylation site (Km approximately 900 microM). p56lck was also found to be highly active for a purified protein-tyrosyl kinase (Vmax greater than 400 pmol.min-1.micrograms-1 using the TCR-zeta (52-164) as a substrate). A variety of agents were tested for their ability to inhibit p56lck, with zinc ions (I50 approximately 1.7 mM) and staurosporine (I50 approximately 500 nM) proving the most potent.     

Phosphorylation of multiple CD3 zeta tyrosine residues leads to formation of pp21 in vitro and in vivo. Structural changes upon T cell receptor stimulation.

T lymphocyte activation resulting from antigen recognition involves a protein tyrosine kinase pathway which triggers phosphorylation of several cellular substrates including the CD3 zeta subunit of the T cell receptor (TCR) to form pp21. The homologous TCR-associated protein, CD3 eta, is an alternatively spliced product of the same gene locus as CD3 zeta. CD3 eta lacks one of six cytoplasmic tyrosine residues (Tyr-132) found in CD3 zeta and is itself not phosphorylated. Site-directed mutagenesis in conjunction with in vitro and in vivo phosphorylation studies herein demonstrates that Tyr-132 is required for the formation of pp21. Moreover, the differential phosphorylation of CD3 zeta versus CD3 eta is not due to a selective association of the known TCR-associated protein tyrosine kinase, p59fyn; p59fyn but not p56lck or p62yes is associated with each of the three TCR isoforms containing CD3 zeta 2, or CD3 eta 2, or CD3 zeta-eta. This association occurs through components of the TCR complex distinct from CD3 zeta or CD3 eta. In addition, we show that pp21 formation is not only dependent on Tyr-132 but results from concomitant phosphorylation of other CD3 zeta residues including Tyr-121. Mutation of Tyr-90, -121, or -132 does not alter primary signal transduction as shown by the ability of individual CD3 zeta Tyr----Phe mutants to produce interleukin-2 upon TCR stimulation. Thus, the substantial structural changes in CD3 zeta upon TCR stimulation as reflected by alteration in its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis may affect subsequent events such as receptor desensitization, receptor movement, and/or protein associations.